Protein expression strains

ABSTRACT

The invention provides an improved host strain for production of desired protein.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Provisional Patent Application No. 62/270,762, filed on Dec. 22, 2015,and of U.S. Provisional Patent Application No. 62/278,728, filed on Jan.14, 2016. This application also claims the benefit of the filing date ofEuropean Patent Application No. 16152977.1, filed on Jan. 27, 2016. Theentire contents of each of the above-referenced applications areincorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in paper and computerreadable form. The paper and computer readable form of the sequencelisting are part of the specification or are otherwise incorporatedherein by reference.

FIELD OF THE INVENTION

This invention relates primarily to the development of fungal strainswhich express proteins at levels substantially higher than the parentalstrains.

BACKGROUND OF THE INVENTION

For some 30 years, desired heterologous proteins have been produced inmicroorganisms. However, having introduced the necessary coding sequenceand obtained expression, much still remains to be done in order tooptimise the process for commercial production. One area of interestconcerns strain improvement, that is to say finding or making strains ofthe host microorganism which enable the protein to be made in higheryields or better purity, for example.

To increase the yield, once a good expression system has been devised,one might envisage trying to increase the copy number of the codingsequence, or to increase the quantity or stability of the mRNA, or toimprove folding and/or secretion of the protein or to decrease thedegradation of the protein. However, the desired effect of increasedexpression will only be seen if the limiting factor(s) is targeted.

Therefore, what is required is a host strain which allows the yield of adesired protein, such as a heterologous protein, to be increased. Theinventors have surprisingly identified that mutation of NOT4 (also knownas MOT2) results in such an increased yield.

Not4 is a ubiquitin-ligating enzyme and is part of the Ccr4-Not complex.The Ccr4-Not complex is conserved in eukaryotic cells, and in yeast thecomplex consists of 9 core subunits: Ccr4, Caf1, Caf40, Caf130, Not1,Not2, Not3, Not4 and Not 5 (Collart, 2003, Global control of geneexpression in yeast by the Ccr4—Not complex. Gene 313: 1-16; Bai et al.,1999, The CCR4 and Caf1 proteins of the Ccr4—Not complex are physicallyand functionally separated from Not2, Not4, and Not5. Mol. Cell. Biol.19: 6642-6651). The complex has been proposed to function as a centralswitchboard that can interpret signals from the environment andcoordinate all levels of gene expression to economically respond to thesignal (Collart, 2012, The Ccr4-Not complex. Gene 492(1): 42-53). It isthought that Not proteins (Not1, Not2, Not3, Not4) are necessary forassembly of the RNA polymerase II complex, which suggests a global rolein transcription regulation (Collart, 1994, Not1(cdc39), Not2(cdc36),Not3, and Not4 encode a global-negative regulator of transcription thatdifferentially affects tata-element utilization. Genes & Development8(5): 525-537; Collart, 2012, as cited above).

Recently a co-crystal structure suggested how the C-terminal region ofNot4 wraps around a HEAT-repeat region of Not1, the scaffold protein inthe Ccr4-Not complex (Bhaskar, 2015, Architecture of the ubiquitylationmodule of the yeast Ccr4-Not complex. Structure 23(5): 921-8).

SUMMARY OF THE INVENTION

The invention provides a fungal host cell having:

-   -   a. a modified Not4 protein or homolog thereof, or    -   b. a modified activity level of Not4 protein or homolog thereof,        or    -   c. a modified NOT4 gene or homolog thereof, or    -   d. a modified level of expression of NOT4 gene or homolog        thereof.

The invention also provides a culture of fungal host cells containing apolynucleotide sequence encoding a desired protein, such as aheterologous protein, characterised in that the fungal host cells have areduced activity level of Not4 protein of homolog thereof.

The invention further provides a method for producing a desired protein,such as a heterologous protein, from a fungal host cell.

The invention provides a method for modifying the production yield of adesired polypeptide from a fungal host cell.

The invention also provides a desired protein, such as a heterologousprotein. Albumin or variant, fragment, and/or fusion thereof is apreferred desired protein.

The invention further provides a composition, such as a pharmaceuticalcomposition, comprising the desired protein.

The invention also provides a method of treating a patient comprisingadministering an effective amount of the composition to the patient.

The invention further provides a method of preparing a fungal host cell.

The invention also provides a Not4 protein or homolog thereof comprisingat least 70% identity to SEQ ID NO: 2 and a mutation at a positioncorresponding to one or more position selected from 426, 427, 428, 429,430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443,444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457,458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469 or 470 of SEQID NO: 2.

The invention further provides a polynucleotide encoding a Not4 variantof the present invention.

Any embodiments described herein, including those described only in theexamples and/or the Preferred Embodiments section, are intended to beable to combine with any other embodiments, unless explicitly disclaimedor the combination is improper.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a Venn diagram showing the classes of and relationship betweentwenty amino acids.

FIG. 2 shows the construction of plasmid pDB5438.

FIG. 3 shows the construction of plasmid pDB2244, “rHA” meansrecombinant human albumin, “FL” is a leader sequence.

FIG. 4 shows the construction of plasmid pDB2305, “m FL” is a leadersequence.

FIG. 5 shows the construction of plasmid pDB3029, “Inv” is a leadersequence.

FIG. 6 shows the construction of plasmid pDB5912, “m FL” is a leadersequence.

FIG. 7 shows the construction of plasmid pDB3936, “m FL” is a leadersequence.

DEFINITIONS

Albumin: The term “albumin” means a protein having the same and/or verysimilar tertiary structure as human serum albumin (HSA) or HSA domainsand has similar properties to HSA or the relevant domains. Similartertiary structures are, for example, the structures of the albuminsfrom the species mentioned under parent albumin. Some of the majorproperties of albumin are i) its ability to regulate plasma volume(oncotic activity), ii) a long plasma half-life of around 19 days±5days, iii) binding to gp60, also known as albondin iv) binding to FcRn,v) ligand-binding, e.g. binding of endogenous molecules such as acidic,lipophilic compounds including billirubin, fatty acids, hemin andthyroxine (see also Table 1 of Kragh-Hansen et al, 2002, Biol. Pharm.Bull. 25, 695, hereby incorporated herein by reference), vi) binding ofsmall organic compounds with acidic or electronegative features e.g.drugs such as warfarin, diazepam, ibuprofen and paclitaxel (see alsoTable 1 of Kragh-Hansen et al, 2002, Biol. Pharm. Bull. 25, 695, herebyincorporated herein by reference). Not all of these properties need tobe fulfilled to characterize a protein or fragment as an albumin. If afragment, for example, does not comprise a domain responsible forbinding of certain ligands or organic compounds the variant of such afragment will not be expected to have these properties either.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding apolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression cassette: The term “expression cassette” means thepolynucleotide encoding a polypeptide and the upstream and downstreamcontrol sequences that provide for its expression.

Expression host: The term “expression host” means any host cell thatexpresses a desired protein, particularly a heterologous protein.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Fragment: The term “fragment” means a polypeptide having one or more(several) amino acids deleted from the amino and/or carboxyl terminus ofa mature polypeptide and/or from an internal region of a maturepolypeptide. Fragments may consist of one uninterrupted sequence derivedfrom a polypeptide or may comprise two or more sequences derived fromdifferent parts of the polypeptide. With respect to albumin, a fragmentmay have a size of more than approximately 20 amino acid residues,preferably more than 30 amino acid residues, more preferred more than 40amino acid residues, more preferred more than 50 amino acid residues,more preferred more than 75 amino acid residues, more preferred morethan 100 amino acid residues, more preferred more than 200 amino acidresidues, more preferred more than 300 amino acid residues, even morepreferred more than 400 amino acid residues and most preferred more than500 amino acid residues. In a preferred embodiment a fragmentcorresponds to one or more of the albumin domains. Preferred albumindomains of the invention are domains having at least 70, 75, 80, 85, 90,95, 96, 97, 98, 99, 99.5% or 100% identity to a HSA domain I consistingof amino acid residues 1 to 194±1 to 15 amino acids of SEQ ID NO: 6; atleast 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5% or 100% identity toHSA domain II consisting of amino acid residues 192 to 387±1 to 15 aminoacids of SEQ ID NO: 6 and at least 70, 75, 80, 85, 90, 95, 96, 97, 98,99, 99.5% or 100% identity to HSA domain III consisting of amino acidresidues 381 to 585±1 to 15 amino acids of SEQ ID NO: 6 or a combinationof one or more (several) of these domains, e.g. domain I and II, domainII and III or domain I and III fused together. No generally acceptedconvention for the exact borders of the albumin domains exists and theoverlap in the above mentioned ranges and the allowance of a varyinglength of plus or minus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15 from amino acids, preferably from 1 to 15 amino acids, morepreferably from 1 to 10 amino acids, most preferably from 1 to 5 aminoacids, at the N-terminal and/or C-terminal of the domains, allowing fora total variance in length of up to 30 amino acids, preferably up to 20amino acids, more preferably up to 10 amino acids for each domainreflects this fact and that there may be some diverging opinions on theamino acid residues in the border between the domains belongs to one orthe other domain. For the same reason it may be possible to findreferences to the amino acid residues of albumin domains that divergefrom the numbers above, however, the skilled person will appreciate howto identify the albumin domains based on the teaching in the literatureand the teaching above. Corresponding domains of non-human albumins canbe identified by alignment with HSA using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 3.0.0 or later, more preferably version5.0.0 or later. The optional parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version ofBLOSUM62) substitution matrix. Alternative alignment tools can also beused, for example MUSCLE as described herein. The domains may also bedefined according to Dockal or Kjeldsen: Dockal et al (The Journal ofBiological Chemistry, 1999, Vol. 274(41): 29303-29310) defines thedomains of HSA as: Domain I: amino acids 1 to 197, Domain II: aminoacids 189 to 385 of SEQ ID NO: 6, Domain III: amino acids 381 to 585 ofSEQ ID NO: 6. Kjeldsen et al (Protein Expression and Purification, 1998,Vol 13: 163-169) defines the domains as: Domain I: amino acids 1 to 192,Domain II: amino acids 193 to 382, Domain III: amino acids 383 to 585.Each domain is itself made up of two homologous subdomains namely 1-105,120-194, 195-291, 316-387, 388-491 and 512-585, with flexibleinter-subdomain linker regions comprising residues Lys106 to Glu119,Glu292 to Val315 and Glu492 to Ala511.

Therefore, in this invention, the following domain definitions arepreferred. The amino acid numbers correspond to those of SEQ ID NO: 6(HSA). However, using these numbers, the skilled person can identifycorresponding domains in other albumin sequences. Domain I may or maynot start at amino acid 1 and may or may not end at any of amino acids192, 193, 194, 195, 196 or 197, preferably any of amino acids 192, 194or 197. Domain II may or may not start at amino acid 189, 190, 191, 192or 193, preferably any of amino acids 189, 192 or 193, and may or maynot end at amino acid 382, 383, 384, 385, 386 or 387, preferably any ofamino acids 382, 285 or 387. Domain III may or may not start at aminoacid 381, 382 or 383, preferably amino acid 381 or 383, and may or maynot end at amino acid 585. Domains in non-human albumins may have thesame or different amino acid lengths and/or residue numbers as HSA. Forexample, a multiple alignment or pair-wise alignment may be preparedusing HSA and one or more (several) other albumins, fragments,derivatives, variants and/or fusions in order to identify domainscorresponding to domains 1, 2 and/or 3 of HSA.

Fusion partner: Throughout this specification, a fusion partner is anon-albumin moiety which may be genetically fused to an albumin orvariant and/or fragment thereof.

Heterologous protein: a heterologous protein is one not naturallyproduced by the host cell and, preferably, does not include proteinssuch as selection markers (e.g. antibiotic resistance markers,auxotrophic selectable markers), chaperones, FLP, REP1, or REP2.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. The mature sequence of humanalbumin is provided in SEQ ID NO: 6, while an example of an immatureform is provided in SEQ ID NO: 8.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptide. Anexample of a mature polypeptide coding sequence of human albumin isprovided in SEQ ID NO: 5, while an example of a coding sequence for animmature form of human albumin is provided in SEQ ID NO: 7.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide, such that the controlsequence directs expression of the coding sequence.

Parent or Parent Albumin: The term “parent” or “parent albumin” means analbumin to which an alteration is made to produce the albumin variantsof the present invention. The parent may be a naturally occurring(wild-type) polypeptide or an allele thereof or a variant thereof. In apreferred embodiment the parent albumin is a wild-type albumin, morepreferably a wild-type albumin from Homo sapiens as disclosed in SEQ IDNO: 8 (UNIPROT: P02768.2) or the mature sequence thereof (SEQ ID NO: 6).Alternative wild-type albumins can be selected the non-exhaustive listshown in Table 1.

TABLE 1 Wild-type albumins from various species. SwissProt or % IdentityGenBank to SEQ Length Common Name Species Accession No ID NO: 6* (aa)Human Homo sapiens P02768.2 100.0 609 Chimpanzee Pan XP_517233 98.8 609troglodytes (predicted sequence) Sumatran Pongo abelii Q5NVH5.2 98.5 609Orangutan Macaque Macaca Q28522.1 93.3 600 (Rhesus mulatta Monkey) CatFelis catus P49064.1 81.9 608 Dog Canis lupus P49822.3 80.0 608familiaris Donkey Equus asinus Q5XLE4.1 76.7 607 Horse Equus caballusP35747.1 76.3 607 Blood Schistosoma Q95VB7 76.2 608 fluke mansoni BovineBos taurus P02769.4 75.6 607 (NP_851335.1) Pig Sus scrofa P08835.2 75.1607 Sheep Ovis aries P14639.1 75.0 607 Goat Capra hircus ACF10391.1 74.8607 Rabbit Oryctolagus P49065.2 74.3 608 cuniculus Mongolian MerionesO35090.1 73.6 609 Gerbil unguiculatus Rat Rattus P02770. 2. 73.3 608norvegicus Mouse Mus musculus P07724.3. 72.3 608 Guinea Pig Caviaporcellus Q6WDN9 72.1 608 Chicken Gallus gallus P19121.2 47.0 615*Sequence identity was calculated using the Needleman-Wunsch algorithmas implemented in the Needle program of EBLOSUM62 (EMBOSS suite ofprograms, version 6.1.0) using gap open penalty of 10, gap extensionpenalty of 0.5 as described herein.

Preferably the parent albumin is a mature albumin. In another embodimentthe parent albumin is at least 70%, more preferably 75%, more preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% at least 99.5% or at least 99.8% identical toSEQ ID NO: 6, and maintains at least one of the major properties ofalbumin or a similar tertiary structure as albumin, such as HSA. Majorproperties of albumin are summarized in Sleep, 2015, “Albumin and itsapplication in drug delivery”, Expert Opinion on Drug Delivery 12(5):793-812.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity.”

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the —nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the —nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Variant: The term “variant” means a polypeptide derived from a parentpolypeptide, e.g. albumin, comprising an alteration, i.e., asubstitution, insertion, and/or deletion, at one or more (several)positions. A substitution means a replacement of an amino acid occupyinga position with a different amino acid; a deletion means removal of anamino acid occupying a position; and an insertion means adding 1-3 aminoacids adjacent to an amino acid occupying a position. The alteredpolypeptide (variant) can be obtained through human intervention bymodification of the polynucleotide sequence encoding the parentalpolypeptide, e.g. albumin. The variant albumin is preferably at least70%, preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5% or at least 99.8% identical to SEQ ID NO: 6and may or may not maintain at least one of the major properties of theparent albumin or a similar tertiary structure such as HSA. Generally,variants or fragments of HSA will have at least 10% (preferably at least50%, 60%, 70%, 80%, 90% or 95%) of HSA ligand binding activity (forexample bilirubin-binding) and at least 50% (preferably at least 70%,80%, 90% or 95%) of HSA's oncotic activity, weight for weight. Oncoticactivity, also known as colloid osmotic pressure, of albumin, albuminvariants or fragments of albumin may be determined by the methoddescribed by Hoefs, J. C. (1992) Hepatology 16:396-403. Bilirubinbinding may be measured by fluorescence enhancement at 527 nm relativeto HSA. Bilirubin (1.0 mg) is dissolved in 50 microL of 1M NaOH anddiluted to 1.0 mL with demineralised water. The bilirubin stock isdiluted in 100 mM Tris-HCl pH8.5, 1 mM EDTA to give 0.6 nmol ofbilirubin/mL in a fluorometer cuvette. Fluorescence is measured byexcitation at 448 nm and emission at 527 nm (10 nm slit widths) duringtitration with HSA over a range of HSA:bilirubin ratios from 0 to 5mol:mol. The variant may have altered binding affinity to FcRn and/or analtered plasma half-life when compared to the parent albumin.

With respect to a variant Not4 protein, the same principles apply, withthe exception that activity is Not4 activity rather than albuminactivity.

The variant polypeptide sequence is preferably one which is not found innature.

Vector: The term “vector” means a linear or circular DNA molecule thatcomprises a polynucleotide encoding a polypeptide and is operably linkedto control sequences that provide for its expression. Vectors includeplasmids. Vectors include expression vectors.

Wild-type: The term “wild-type” (WT) albumin means an albumin having thesame amino acid sequence as the albumins naturally found in an animal orin a human being. SEQ ID NO: 6 is an example of a wild-type albumin fromHomo sapiens. The “wild-type” (WT) human albumin (HSA) sequence is givenby GenBank Accession number AAA98797.1 (Minghetti et al. “Molecularstructure of the human albumin gene is revealed by nucleotide sequencewithin q11-22 of chromosome 4”, J. Biol. Chem. 261 (15), 6747-6757(1986)). Examples of wild-type albumins are provided in Table 1 (above).

Conventions for Designation of Amino Acid Positions

For purposes of the present invention, the polypeptide disclosed in SEQID NO: 2 is used to determine the corresponding amino acid residue in ahomolog of Not4 protein. The amino acid sequence of a homolog of Not4protein is aligned with the polypeptide disclosed in SEQ ID NO: 2, andbased on the alignment, the amino acid position number corresponding toany amino acid residue in the polypeptide disclosed in SEQ ID NO: 2 isdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofthe EMBOSS package (EMBOSS: The European Molecular Biology Open SoftwareSuite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version5.0.0 or later. The parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix.

Identification of the corresponding amino acid residue in a homolog ofNot4 protein can be determined by an alignment of multiple polypeptidesequences using several computer programs including, but not limited to,MUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010,Bioinformatics 26: 1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

In describing the polypeptides of the present invention, thenomenclature described below is adapted for ease of reference. Theaccepted IUPAC single letter or three letter amino acid abbreviation isemployed.

Substitutions. For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of threonine at position 226 withalanine is designated as “Thr226Ala” or “T226A”. Multiple mutations areseparated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or“G205R+5411 F”, representing substitutions at positions 205 and 411 ofglycine (G) with arginine (R) and serine (S) with phenylalanine (F),respectively.

Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofglycine at position 195 is designated as “Gly195*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*”or “G195*+S411*”.

Insertions. As disclosed above, an insertion may be to the N-side(‘upstream’, ‘X−1’) or C-side (‘downstream’, ‘X+1’) of the amino acidoccupying a position (‘the named (or original) amino acid’, ‘X’).

For an amino acid insertion to the C-side (‘downstream’, ‘X+1’) of theoriginal amino acid (‘X’), the following nomenclature is used: Originalamino acid, position, original amino acid, inserted amino acid.Accordingly, the insertion of lysine after glycine at position 195 isdesignated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

For an amino acid insertion to the N-side (‘upstream’, ‘X−1’) of theoriginal amino acid (X), the following nomenclature is used: Originalamino acid, position, inserted amino acid, original amino acid.Accordingly, the insertion of lysine (K) before glycine (G) at position195 is designated “Gly195LysGly” or “G195KG”. An insertion of multipleamino acids is designated [Original amino acid, position, inserted aminoacid #1, inserted amino acid #2; etc., original amino acid]. Forexample, the insertion of lysine (K) and alanine (A) before glycine atposition 195 is indicated as “Gly195LysAlaGly” or “G195KAG”. In suchcases the inserted amino acid residue(s) are numbered by the addition oflower case letters with prime to the position number of the amino acidresidue following the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195a′ 195b′ 195 G K - A - G

Multiple alterations. Polypeptides comprising multiple alterations areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of arginine and glycine atpositions 170 and 195 with tyrosine and glutamic acid, respectively.

Different alterations. Where different alterations can be introduced ata position, the different alterations are separated by a comma, e.g.,“Arg170Tyr,Glu” represents a substitution of arginine at position 170with tyrosine or glutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala”designates the following variants:

“Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and“Tyr167Ala+Arg170Ala”.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the invention provides a fungal host cell having:

-   -   a. a modified Not4 protein or homolog thereof, or    -   b. a modified level of activity of Not4 protein or homolog        thereof, or    -   c. a modified NOT4 gene or homolog thereof, or    -   d. a modified level of expression of NOT4 gene or homolog        thereof.

NOT4 is also known as MOT2. The modified Not4 protein may be modifiedrelative to a reference Not4 protein such as a wild-type Not4 proteinfor example SEQ ID NO: 2. Preferably, the modified Not4 protein orhomolog thereof has at least 70% identity to SEQ ID NO: 2, morepreferably at least 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.1, 99.2,99.3, 99.4, 99.5, 99.6, 99.7, 99.8 or at least 99.9% identity to SEQ IDNO: 2. More preferably, the modified Not4 protein comprises or consistsof SEQ ID NO: 4.

It is preferred that the modified level of Not4 protein or homologthereof is a reduced expression level of Not4 protein or homolog thereofor a reduced activity level of Not4 protein or homolog thereof.Preferably the reduced level is relative to the level in a referencefungal host cell, such as a fungal host cell in which the Not4 proteincomprises or consists of SEQ ID NO: 2. The Not4 protein of the referencefungal host may be a wild-type Not4 sequence, such as SEQ ID NO: 2. Asuitable reference fungal host cell is S. cerevisiae S288C or S.cerevisiae DXY1. S288C has the genotype MATα SUC2 gal2 mal2 mel flo1flo8-1 hap1 ho bio1 bio6. DXY1 has the genotype leu2-3, leu2-122, can1,pra1, ubc4, ura3:yap3 (Kerry-Williams et al. (1998) Yeast 14:161-169).Other suitable reference fungal host cells include cells which areidentical to the host cell with the exception of the NOT4 gene or Not4protein or homolog thereof. For example, the NOT4 gene of the referencemay be wild-type (e.g. SEQ ID NO: 1) or the NOT4 gene of the referencemay encode wild-type Not4 protein (e.g. SEQ ID NO: 2) or the Not4protein encoded by the reference may be wild-type (e.g. SEQ ID NO: 2).Preferably, the host cell of the invention is identical to a parentstrain with the exception of the NOT4 gene or Not4 protein or homologthereof. A reference fungal host may also be referred to as a“corresponding” fungal host. A reference fungal host may be a parentfungal host.

A reduced level of Not4 protein or activity level of Not4 protein may beachieved, for example, by mutating or deleting the NOT4 gene, thusresulting a mutated Not4 protein or homolog thereof or complete absenceof Not4 protein or homolog thereof; by removing or changing the openreading frame of the gene, by mutating or changing control sequences ofthe NOT4 gene such as a promoter sequence and/or a terminator sequence;by blocking or reducing transcription of the NOT4 gene for example byintroducing suitable interfering RNA such as antisense mRNA, byintroducing, controlling or modifying suitable transcriptional activatorgenes or by introducing an agent which blocks activity level of Not4protein or homolog thereof. Methods of measuring protein levels andprotein activity are well known in the art.

The modified activity level of the Not4 protein or homolog thereof maybe reduced, therefore resulting in from 0, 10, 20, 30, 40, 50, 60, 70,80 or 90 to 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95% of the activitylevel of Not4 protein or homolog thereof of a parent or reference fungalhost cell, such as a wild-type fungal host cell. The reduced activitylevel of Not4 protein or homolog thereof in a fungal host cell may berelative to the activity level of Not4 protein or homolog thereof of areference fungal host cell such as a parent fungal host cell or awild-type fungal host cell as described above. Consequently, theactivity level of Not4 protein or homolog thereof in the host cell is atmost 95% of the activity level of Not4 protein or homolog thereof in areference fungal host cell, for example at most 90, 80, 70, 60, 50, 40,30, 20, or at most 10% of the activity level of Not4 protein or homologthereof in the reference fungal host cell. The activity level of Not4protein or homolog thereof may be reduced to zero or substantially zero.

The modified expression level (amount) of Not4 protein or homologthereof may be reduced, therefore resulting in from 0, 10, 20, 30, 40,50, 60, 70, 80 or 90 to 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95% of theexpression level of Not4 protein or homolog thereof of the referencefungal host cell, such as a wild-type fungal host cell. The reducedexpression level of Not4 protein or homolog thereof in a fungal hostcell may be relative to the expression level of Not4 protein or homologthereof of a reference fungal host cell such as a parent fungal hostcell or a wild-type fungal host cell as described above. Consequently,the expression level of Not4 protein or homolog thereof in the host cellis at most 95% of the expression level of Not4 protein or homologthereof in a reference fungal host cell, for example at most 90, 80, 70,60, 50, 40, 30, 20, or at most 10% of the expression level of Not4protein or homolog thereof in the reference fungal host cell. Theexpression level of Not4 protein or homolog thereof may be reduced tozero or substantially zero.

The fungal host cell may lack a functional NOT4 gene or homolog thereofor Not4 protein or homolog thereof. For example, the fungal host cellmay contain a modified NOT4 gene which may result in a reducedexpression level of Not4 protein or homolog thereof, or in reducedactivity level of Not4 protein or homolog thereof. The fungal host cellmay lack a NOT4 gene or homolog thereof, for example due to deletion,and/or may lack Not4 protein or homolog thereof.

The modified Not4 protein, or homolog thereof, may be mutated so thatits interaction with Not1 protein, or homolog thereof, is altered. Forexample, the N-terminal region of Not4 protein, or homolog thereof, maybe mutated, such as the α-helix containing amino acids corresponding topositions 426 to 439 of SEQ ID NO: 2.

Therefore, the invention also provides a fungal host cell having a Not4protein or homolog thereof which has a weaker interaction, such ashydrophobic interaction, with Not1 than the interaction between awild-type Not4 protein (e.g. SEQ ID NO: 2) and a wild-type Not1 protein(e.g. SEQ ID NO: 9).

The fungal host cell may have a modified Not4 protein or homolog thereofcomprising a mutation at a position corresponding to a position selectedfrom 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438,439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452,453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466,467, 468, 469 or 470 of SEQ ID NO: 2, preferably a position selectedfrom:

-   -   a position corresponding to 426, 427, 428, 429, 430, 431, 432,        433, 434, 435, 436, 437, 438, or 439 of SEQ ID NO: 2, preferably        429, 430, 434, or 437, most preferably position 429;    -   a position corresponding to 460, 461, 462, 463, 464, 465, 466,        467, 468, 469 or 470 of SEQ ID NO: 2, preferably 463, 464, or        466; or    -   a position corresponding to 438, 439, 440, 441, 442, 443, 444,        445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, or 456 of        SEQ ID NO: 2, preferably 442, 445, 447 or 452.    -   The mutation may be a substitution, insertion and/or deletion at        one or more (e.g. several) positions. Substitutions are        preferred.

The fungal host cell may comprise a polynucleotide sequence encoding themodified Not4 protein or homolog thereof, for example SEQ ID NO: 3. Dueto the degeneracy of the genetic code, other polynucleotide sequencescan also encode suitable modified Not4 proteins or homologs thereof.

The fungal host cell may comprise a modified Not4 protein or homologthereof in which, relative to SEQ ID NO: 2, the mutation is asubstitution to an amino acid, preferably a non-conserved amino acid,selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Wand Y.

Amino acids fall into various well known classes. Therefore, some aminoacids are more closely related than others. As used herein,“conservative amino acid substitutions” refers to substitutions madewithin the same group, and which typically do not substantially affectprotein function. By “conservative substitution” is intended withingroups such as those shown by FIG. 1., this is a Venn diagram whichprovides one system by which conservation level can be visualized.Generally, substitutions of low conservation are those for which thereare many boundaries (lines) between the starting amino acid and theresultant substitution. “Conservative amino acid substitution” includesa substitution made within the same group such as within:

-   -   aromatic amino acids: F, H, W, Y;    -   aliphatic amino acids: I, L, V;    -   hydrophobic amino acids: A, C, F, H, I, K, L, M, T, V, W, Y;    -   charged amino acids: D, E, H, K, R, for example:        -   positively charged amino acids: H, K, R; or        -   negatively charged amino acids: D, E;    -   polar amino acids: C, D, E, H, K, N, Q, R, S, T, W, Y;    -   small amino acids: A, C, D, G, N, P, S, T, V, for example:        -   tiny amino acids: A, C, G, S.

Alternatively, “conservative substitution” may be within the followinggroups:

-   -   amino acids having aliphatic side chains: G, A, V, L, I;    -   amino acids having aromatic side chains: F, Y, W;    -   amino acids having sulphur-containing side chains: C, M;    -   amino acids having aliphatic hydroxyl side chains: S, T;    -   amino acids having basic side chains: K, R, H;    -   acidic amino acids and their amide derivatives: D, E, N, Q.

Substitutions may be made by techniques known in the art, such as bysite-directed mutagenesis as disclosed in U.S. Pat. No. 4,302,386(incorporated herein by reference).

Non-conservative amino substitutions may refer to substitutions madefrom one group to another group for example from the group havingaromatic side chains to the group having aliphatic side chains.

The mutation at a position corresponding to position 429 of SEQ ID NO: 2may be a substitution from the native amino acid, such as F, to anon-native amino acid such as A, C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y, preferably to G, A, V, L, or I, more preferably toV, L or I, most preferably to I. The substitution may be to anon-conserved amino acid. The substitution may be to an aliphatic aminoacid. A particularly preferred substitution is from F to I.

The mutation at a position corresponding to position 430 of SEQ ID NO: 2may be a substitution from the native amino acid, such as L, to anynon-native amino acid such as A, C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y. The substitution may be to a non-conserved aminoacid.

The mutation at a position corresponding to position 434 may be asubstitution from the native amino acid, such as L, to any non-nativeamino acid such as A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V,W, or Y. The substitution may be to a non-conserved amino acid.

The mutation at a position corresponding to position 437 of SEQ ID NO: 2may be a substitution from the native amino acid, such as L, to anynon-native amino acid such as A, C, D, E, F, G, H, I, K, L, M, N, P, Q,R, S, T, V, W, or Y. The substitution may be to a non-conserved aminoacid.

A preferred modified Not4 protein includes a mutation at a positioncorresponding to F429 of SEQ ID NO: 2.

A preferred modified Not4 protein comprises or consists of SEQ ID NO: 4,i.e. which includes the mutation F429L.

Alternatively, the modified level may be increased. An increased levelor increased activity of Not4 protein or homolog thereof is likely todecrease the yield of desired protein (such as a heterologous protein).Such a decreased yield may be desirable when, for example, the desiredprotein is detrimental to the viability of the host cell. An increasedlevel may be at least 105, 110, 120, 130, 140, 150, 175, or 200% of thelevel in a reference host such as a parent host.

The fungal host cell may be a recombinant fungal host cell.

The fungal host cell may be a yeast or a filamentous fungus. “Fungi” asused herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra).

In a preferred aspect, the fungal host cell is a yeast cell. “Yeast” asused herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo: 9, 1980).

In a more preferred aspect, the yeast host cell is a Candida, Hansenula,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiacell.

In a more preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, Saccharomyces oviformis, Kluyveromyces lactis or a Yarrowialipolytica cell. A Saccharomyces cerevisiae host is particularlypreferred.

The S. cerevisiae host may or may not comprise one or more of thefollowing genotypic features: leu2-3, leu2-122, can1, pra1, ubc4, ura3,yap3::URA3, lys2, hsp150::LYS2, pmt1::URA3 (as defined in WO2014/138371,incorporated herein by reference), for example S. cerevisiae BXP10.Preferably the S. cerevisiae host includes MATa.

The S. cerevisiae host may or may not comprise one or more of thefollowing genotype, MATa, leu2-3, leu2-112, ubc4, ura3, yap3::URA3,lys2, hsp150::LYS2; with PDI1, URA3 and Ylplac211 integrated at the PDI1locus (Finnis et al 2010, Microbial Cell Factories 9:87), for example S.cerevisiae DP9.

The S. cerevisiae host may or may not comprise one or more of thefollowing genotype, MATa, leu2, pep4-3, for example S. cerevisiaeMT302/28B as described in Finnis et al 1993, Eur. J. Biochem, 212:201-210.

The S. cerevisiae host may or may not comprise the following genotype:MATa, SUC2, gal2, mal2, mel, flo1, flo8-1, hap1, ho, bio1, bio6(Mortimer and Johnston (1986) Genetics 113:35-43), for example S.cerevisiae S288C.

A preferred S. cerevisiae host strain comprises or consists of all ofMATa, leu2-3, leu2-122, can1, pra1, ubc4, ura3, yap3::URA3, lys2,hsp150::LYS2, and pmt1::URA3.

Another preferred S. cerevisiae host comprises or consists of all of:MATa, leu2-3, leu2-112, ubc4, ura3, yap3::URA3, lys2, hsp150::LYS2, withPDI1, URA3 and Ylplac211 integrated at the PDI1 locus.

Another preferred S. cerevisiae host comprises or consists of all of:MATa, SUC2, gal2, mal2, mel, flo1, flo8-1, hap1, ho, bio1, bio6.

Another preferred S. cerevisiae host comprises or consists of all of:MATa, leu2, pep4-3.

The host may be polyploid, diploid or halpoid. A haploid or diploidyeast host is preferred, preferably haploid.

The host mating type may be, for example, MATa or MATα (Mat-alpha).Preferably the S. cerevisiae host contains a plasmid encoding humanalbumin or variant, fragment and/or fusion thereof.

“Filamentous fungi” include all filamentous forms of the subdivisionEumycota and Oomycota (as defined by Hawksworth et al., 1995, supra).The filamentous fungi are characterized by a mycelial wall composed ofchitin, cellulose, glucan, chitosan, mannan, and other complexpolysaccharides. Vegetative growth is by hyphal elongation and carboncatabolism is obligately aerobic. In contrast, vegetative growth byyeasts such as Saccharomyces cerevisiae is by budding of a unicellularthallus and carbon catabolism may be fermentative.

Preferred filamentous fungal host cells may or may not includeAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium,Humicola, Magnaporthe, Mucor, Myceliophthora, Neocaffimastix,Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia,Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus,Thielavia, Tolypocladium, Trametes or Trichoderma.

The fungal host cell, may comprise a nucleotide sequence encoding adesired protein. Preferably, the desired protein is a heterologousprotein. A heterologous protein is one not naturally produced by thehost cell and, preferably, does not include proteins such as selectablemarkers, for example antibiotic resistance markers or auxotrophicmarkers, chaperones, FLP or FRT.

The fungal host cell may be an expression host. The fungal host cell maycomprise an expression cassette for example encoding a desired proteinsuch as a heterologous protein. The expression cassette may be, forexample within a vector such as a plasmid. The fungal host cell maycomprise an expression vector.

The desired protein may or not be a plant or animal protein or variantthereof. The desired protein may, or may not, comprise the sequence ofalbumin, a monoclonal antibody, an etoposide, a serum protein (such as ablood clotting factor), antistasin, a tick anticoagulant peptide,transferrin, lactoferrin, endostatin, angiostatin, collagens,immunoglobulins or immunoglobulin-based molecules or fragment of either(e.g. a Small Modular ImmunoPharmaceutical™ (“SMIP”) or dAb, Fab′fragments, F(ab′)2, scAb, scFv or scFv fragment), a Kunitz domainprotein (such as those described in WO03/066824, with or without albuminfusions), interferons, interleukins, IL-10, IL-11, IL-2, interferon α(alpha) species and sub-species, interferon β (beta) species andsub-species, interferon γ (gamma) species and sub-species, leptin, CNTF,CNTF_(A×15), IL-1-receptor antagonist, erythropoietin (EPO) and EPOmimics, thrombopoietin (TPO) and TPO mimics, prosaptide, cyanovirin-N,5-helix, T20 peptide, T1249 peptide, HIV gp41, HIV gp120, urokinase,prourokinase, tPA, hirudin, platelet derived growth factor, parathyroidhormone, proinsulin, insulin, glucagon, glucagon-like peptides such asexendin-4, GLP-1 or GLP-2, insulin-like growth factor, calcitonin,growth hormone, transforming growth factor β (beta), tumour necrosisfactor, G-CSF, GM-CSF, M-CSF, FGF, coagulation factors in both pre andactive forms, including but not limited to plasminogen, fibrinogen,thrombin, pre-thrombin, pro-thrombin, von Willebrand's factor,alpha₁-antitrypsin, plasminogen activators, Factor VII, Factor VIII,Factor IX, Factor X and Factor XIII, nerve growth factor, LACI,platelet-derived endothelial cell growth factor (PD-ECGF), glucoseoxidase, serum cholinesterase, aprotinin, amyloid precursor protein,inter-alpha trypsin inhibitor, antithrombin III, apo-lipoproteinspecies, Protein C, Protein S, a metabolite, an antibiotic, or a variantor fragment of any of the above.

Preferably the variant has at least 70, 75, 80, 85, 90, 91, 92, 93, 94,95, 96, 97, 98, or 99% identity to one or more of the proteins disclosedabove.

A preferred desired protein may or may not be a serum protein such as analbumin or variant, fragment and/or fusion thereof. Preferably, thealbumin has from 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 98.2, 98.4,98.6, 98.8, 99, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9 to 70,75, 80, 85, 90, 95, 96, 97, 98, 98.2, 98.4, 98.6, 98.8, 99, 99.2, 99.3,99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100% sequence identity to SEQ IDNO: 6. Most preferably, the albumin comprises or consists of SEQ ID NO:6.

The albumin variant, fragment and/or fusion thereof may or may notcomprise A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y at aposition corresponding to position K573 of SEQ ID NO: 6, more preferablya P, H, W or Y at a position corresponding to position K573 of SEQ IDNO: 6. Particularly preferred albumin variants have at least 95%identity to SEQ ID NO: 6 (more preferably at least 96, 97, 98 or 99%identity) and comprise P at a position corresponding to 573 of SEQ IDNO: 6.

Other preferred albumin variants, fragments and/or fusions thereofinclude those disclosed in WO2011/051489, WO2011/124718, WO2012/059486,WO2012/150319, WO2014/072481, WO2013/135896, WO2015/036579,WO2010/092135, WO2013/075066, WO2014/179657, WO2009/126920,WO2010/059315, WO2011/103076, WO2012/112188 and WO2015/063611 orfragments of fusions thereof (each incorporated herein by reference).

The albumin may or may not be a fragment of an albumin or variantthereof.

The albumin variant, fragment and/or fusion thereof may have a bindingaffinity to FcRn that is stronger or weaker (and, preferably, isstronger) than that of the parent albumin, fragment and/or fusionthereof.

The albumin variant, fragment and/or fusion thereof may have a KD toFcRn (e.g. shFcRn) that is lower than the corresponding KD for HSA orconjugate thereof to. Preferably, the KD for the albumin variant,fragment and/or fusion thereof is less than 0.9×KD for HSA to FcRn, morepreferred less than 0.5×KD for HSA to FcRn, more preferred less than0.1×KD for HSA to FcRn, even more preferred less than 0.05×KD for HSA toFcRn, even more preferred less than 0.02×KD for HSA to FcRn, even morepreferred less than 0.01×KD for HSA to FcRn and most preferred less than0.001×KD for HSA to FcRn (where X means ‘multiplied by’). A lower KDcorresponds to a stronger binding affinity.

The albumin variant, fragment and/or fusion thereof may have a KD toFcRn that is higher than the corresponding KD for HSA or conjugatethereof to FcRn. Preferably, the KD for the albumin variant, fragmentand/or fusion thereof is more than 2×KD for HSA to FcRn, more preferredmore than 5×KD for HSA to FcRn, more preferred more than 10×KD for HSAto FcRn, even more preferred more than 25×KD for HSA to FcRn, mostpreferred more than 50× KD for HSA to FcRn. The albumin variant,fragment and/or fusion thereof may be a null binder to FcRn. A higher KDcorresponds to a weaker binding affinity.

When determining and/or comparing KD, one or more (e.g. several) (andpreferably all) of the following parameters may be used:

Instrument: Biacore 3000 instrument (GE Healthcare)

Flow cell: CM5 sensor chip

FcRn: human FcRn, preferably soluble human FcRn, optionally coupled to atag such as Glutathione S Transferase (GST) or Histidine (His), mostpreferably His such as 6 histidine residues at the C-terminus of thebeta-2-microglobulin.

Quantity of FcRn: 1200-2500 RU

Coupling chemistry: amine coupling chemistry (e.g. as described in theprotocol provided by the manufacturer of the instrument).

Coupling method: The coupling may be performed by injecting 20 μg/ml ofthe protein in 10 mM sodium acetate pH 5.0 (GE Healthcare). Phosphatebuffer (67 mM phosphate buffer, 0.15 M NaCl, 0.005% Tween 20) at pH 5.5may be used as running buffer and dilution buffer. Regeneration of thesurfaces may be done using injections of HBS-EP buffer (0.01 M HEPES,0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20) at pH 7.4 (Biacore AB).

Quantity of injection of test molecule (e.g. HSA or variant) 20-0.032μM.

Flow rate of injection: constant, e.g. 30 μl/ml.

Temperature of injection: 25° C.

Data evaluation software: BIAevaluation 4.1 software (BIAcore AB).

The albumin variant, fragment and/or fusion thereof may have a plasmahalf-life that is longer or shorter, preferably longer, than that of theparent albumin, fragment and/or fusion thereof.

Plasma half-life is ideally determined in vivo in suitable individuals.However, since it is time consuming and expensive and inevitably thereare ethical concerns connected with doing experiments in animals or man,it is desirable to use an in vitro assay for determining whether plasmahalf-life is extended or reduced. It is known that the binding ofalbumin to its receptor (FcRn) is important for plasma half-life and thecorrelation between receptor binding and plasma half-life is that ahigher affinity of albumin to its receptor leads to longer plasmahalf-life. Thus, for the invention, a higher affinity of albumin to FcRnis considered indicative of an increased plasma half-life and a loweraffinity of albumin to its receptor is considered indicative of areduced plasma half-life.

The binding of albumin to its receptor FcRn may be described using theterm affinity and the expressions “stronger” or “weaker”. Thus, itshould be understood that a molecule having a higher affinity to FcRnthan HSA is considered to bind more strongly to FcRn than HSA and amolecule having a lower affinity to FcRn than HSA is considered to bindmore weakly to FcRn than HSA. The term ‘binding coefficient’ can be usedinstead of the term ‘binding affinity’.

The terms “longer plasma half-life” or “shorter plasma half-life” andsimilar expressions are understood to be in relationship to thecorresponding parent or reference or corresponding albumin molecule.Thus, a longer plasma half-life with respect to a variant albumin of theinvention means that the variant has longer plasma half-life than thatof the corresponding albumin having the same sequences except for thealteration(s) described herein.

The albumin or variant, and/or fragment thereof may or may not begenetically fused to a fusion partner. Preferably, the fusion partner isa non-albumin protein. The fusion partner may be fused at the N′ or C′terminus of the albumin. There may or may not be one or more spaceramino acids located between the albumin moiety and the partner moiety.Fusion partners may be inserted within the albumin sequence. The fusionpartner may be at least 5 amino acids long, for example at least 5, 10,15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or at least 100 amino acidslong. The fusion partner may or may not have a maximum length of from35, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 300, 400, 500,600, 700, 800, 900 or 1000 amino acids long. The fusion protein maycomprise one or more fusion partners, for example fused at the N′ or C′terminus of albumin or inserted within the albumin sequence. The fusionprotein may comprise one or more (e.g. several, such as 2, 3, 4 or 5)copies of the same fusion partner or two or more different partners. Thefusion partner may be selected from desired or heterologous proteins asdisclosed above.

A preferred fusion protein may comprise a polypeptide having GLP-1activity such as those described in WO2014/138371 (incorporated hereinby reference, with particular reference to pages 13, 14, 26, 34 to 37).For example, a preferred fusion protein may comprise HSA (SEQ ID NO: 6),or a variant and/or fragment of HSA genetically fused in series to onecopy of a GLP analog (e.g. SEQ ID NO: 10) or HSA (SEQ ID NO: 6), or avariant and/or fragment of HSA genetically fused in series to a tandemrepeat of a GLP analog (e.g. SEQ ID NO: 11). For example, the fusionprotein may comprise or consist of SEQ ID NO: 12 (albiglutide).

Particularly suitable fungal host cells for the production of albumins,variants, fragments and/or fusions thereof include, but are not limitedto, Aspergillus (WO06/066595), Kluyveromyces (Fleer, 1991,Bio/technology 9: 968-975), Pichia (Kobayashi, 1998, TherapeuticApheresis 2: 257-262) and Saccharomyces (Sleep, 1990, Bio/technology 8:42-46)), each incorporated herein by reference.

The desired protein (such as a heterologous protein) may or may not be asecreted protein. Therefore, the protein encoded by the host cell may ormay not comprise a signal peptide (which in some literature may also bereferred to as a “leader sequence”). Typically, the signal peptidesequence is cleaved from the protein during secretion from the hostcell, therefore the resultant (mature) protein does not comprise asignal peptide sequence. Examples of suitable signal peptide sequencesare provided below. A signal peptide may or may not comprise apropeptide.

Alternatively, the desired protein may or may not be intracellular.

The desired protein may or may not be encoded by a plasmid.

The desired protein may or may not be encoded by chromosomal nucleicacid.

Suitable plasmids include 2 micron family plasmids such as thosedescribed in WO2006/067511 (incorporated herein by reference, withparticular emphasis on the section titled “The 2 μm-family plasmids:” onpages 46 to 61). Such plasmids, collectively termed “2 μm-familyplasmids”, include pSR1, pSB3 and pSB4 from Zygosaccharomyces rouxii(formerly classified as Zygosaccharomyces bisporus), plasmids pSB1 andpSB2 from Zygosaccharomyces bailii, plasmid pSM1 from Zygosaccharomycesfermentati, plasmid pKD1 from Kluyveromyces drosphilarum, an un-namedplasmid from Pichia membranaefaciens (“pPM1”) and the 2 μm plasmid (suchas shown in FIG. 1 of WO2006/067511) and variants (such as Scp1, Scp2and Scp3) from Saccharomyces cerevisiae (Volkert, et al., 1989,Microbiological Reviews 53: 299; Murray et al., 1988, J. Mol. Biol. 200:601; Painting, et al., 1984, J. Applied Bacteriology 56: 331).

A 2 μm-family plasmid typically comprises at least three open readingframes (“ORFs”) that each encodes a protein that functions in the stablemaintenance of the 2 μm-family plasmid as a multicopy plasmid. Theproteins encoded by the three ORFs can be designated FLP, REP1 and REP2.Where a 2 μm-family plasmid comprises not all three of the ORFs encodingFLP, REP1 and REP2 then ORFs encoding the missing protein(s) should besupplied in trans, either on another plasmid or by chromosomalintegration.

A preferred plasmid is the 2 μm plasmid from S. cerevisiae, preferablyencoding a desired protein such as a heterologous protein.

The Not4 protein and/or the desired, e.g. heterologous, protein may beencoded by a nucleotide sequence operably linked to one or more controlsequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a polypeptide. Manipulation of the polynucleotideprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotides utilizing recombinant DNA methods are known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the polypeptide. The promoter may be any polynucleotidethat shows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO96/00787), Fusariumvenenatum amyloglucosidase (WO00/56900), Fusarium venenatum Daria(WO00/56900), Fusarium venenatum Quinn (WO00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, supra.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the polypeptide. Any terminator that is functional in the hostcell may be used.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leadersequence is operably linked to the 5′-terminus of the polynucleotideencoding the polypeptide. Any leader that is functional in the host cellmay be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polypeptide-encoding sequenceand, when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Preferred signal peptides for yeast host cells, for example yeast hostcells for the production of albumin, or variant, fragment and/or fusionthereof, include:

a signal peptide obtained from the gene for Saccharomyces cerevisiaealpha-factor,

a signal peptide obtained from the gene for Saccharomyces cerevisiaeinvertase,

a signal peptide obtained from the gene for Saccharomyces cerevisiaeKEX2 e.g. comprising or consisting of SEQ ID NO: 13 or a modified KEX2signal peptide sequence e.g. comprising or consisting of SEQ ID NO: 14.

Particularly preferred signal peptides include:

a signal peptide comprising a fusion of the mating factor alpha signalpeptide sequence and the human albumin signal peptide sequence as taughtin WO90/01063 (incorporated herein by reference), an example of such asignal peptide sequence is provided in SEQ ID NO: 15;

a signal peptide comprising the pentapeptide motif of SEQ ID NO: 16,wherein the pentapeptide motif is located in the hydrophobic domain ofthe signal peptide sequence, for example from positions −10 to −25 of animmature protein, where position −1 refers to the amino acid of thesignal peptide sequence which is immediately adjacent the N-terminus ofthe first amino acid of the mature sequence, or for signal peptidesequences comprising a propeptide position −1 refers to the amino acidof the signal peptide sequence which is immediately adjacent theN-terminus of the first amino acid of the propeptide, examples of suchsignal peptide sequences are disclosed in WO2004/009819 (incorporatedherein by reference);

an albumin signal peptide which is modified to comprise the pentapeptidemotif of SEQ ID NO: 16, the pentapeptide motif may be located in thehydrophobic domain of the signal peptide sequence, an example of such amodified signal peptide sequence is provided in SEQ ID NO: 17. Thepentapeptide motif may be inserted into an invertase signal peptide togenerate a modified invertase signal peptide, examples of modifiedinvertase signal peptides are provided in SEQ ID NO: 35 and SEQ ID NO:36; or an albumin signal peptide which is modified to comprise thepentapeptide motif of SEQ ID NO: 16 and comprises a propeptide at the C′terminus of the signal peptide sequence, the pentapeptide motif may belocated in the hydrophobic domain of the signal peptide sequence,examples of such a modified signal peptide sequence are provided in SEQID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.

Signal peptides comprising of or consisting of SEQ ID NO: 15, SEQ ID NO:20 and SEQ ID NO: 36 are especially preferred, for example forexpression of albumin or a variant, fragment and/or fusion thereof.

Other useful signal peptide coding sequences are described by Romanos etal., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Saccharomycescerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of thepolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. In yeast, theADH2 system or GAL1 system may be used. In filamentous fungi, theAspergillus niger glucoamylase promoter, Aspergillus oryzae TAKAalpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter maybe used. Other examples of regulatory sequences are those that allow forgene amplification. In eukaryotic systems, these regulatory sequencesinclude the dihydrofolate reductase gene that is amplified in thepresence of methotrexate, and the metallothionein genes that areamplified with heavy metals. In these cases, the polynucleotide encodingthe polypeptide would be operably linked with the regulatory sequence.

The host strain may or may not express or overexpress one or morechaperone proteins such as those described in WO2005/061718,WO2006/067511, WO2006/136831 or WO2014/138371, all incorporated hereinby reference. For example, the host strain may or may not overexpressone or more of: AHA1, CCT2, CCT3, CCT4, CCT5, CCT6, CCT7, CCT8, CNS1,CPR3, CPRE, ER01, EUG1, FM01, HCH1, HSP10, HSP12, HSP104, HSP26, HSP30,HSP42, HSP60, HSP78, HSP82, JEM1, MDJ1, MDJ2, MPD1, MPD2, PDI1, PFD1,ABC1, APJ1, ATP11, ATP12, BTT1, CDC37, CPR7, HSC82, KAR2, LHS1, MGE1,MRS11, NOB1, ECM10, SSA1, SSA2, SSA3, SSA4, SSC1, SSE2, SIL1, SLS1,ORM1, ORM2, PER1, PTC2, PSE1, UBI4 and HAC1 or a truncated intronlessHAC1 (Valkonen et al., 2003, Applied Environ. Micro., 69: 2065), as wellas T/M9, PAM18 (also known as TIM14) and TCP1 (also known as CCT1) or avariant thereof. Overexpression of PDI1 (SEQ ID NO: 21) or variant orfragment thereof and/or ERO1 (SEQ ID NO: 22) or variant or fragmentthereof is preferred. Over-expression includes increasing the expressionof the chaperone by at least 25, 50, 75, 100, 200, 300, 400, 500%relative to the native level expression of the chaperone in the hostcell. Over-expression may correspond to an increase in chaperone amount,or an increase in chaperone activity. Overexpression may be achieved byincreasing the copy number of the gene encoding the chaperone, forexample by providing a host cell comprising 2, 3, 4, 5, 6, 7, 8, 9, 10or more copies of the gene. Preferably the variant chaperone has atleast 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequenceidentity to the chaperone. Preferably the variant maintains thefunctional activity of the chaperone.

The host cell may or may not comprise at least one heterologous nucleicacid encoding a protease or a fragment and/or variant thereof. The hostcell may or may not comprise at least one nucleic acid encoding aprotease such as a calcium dependent serine protease such as killerexpression protease (Kex2p) or a fragment and/or variant thereof.Preferably the protease variant or fragment is functional, for examplehave the ability to cleave polypeptides at the carboxyl end of therecognition sequence Arg-Arg/X or Lys-Arg/X. A KEX2 nucleotide sequencemay comprise or consist of SEQ ID NO: 23, a Kex2p protein may compriseor consist of SEQ ID NO: 24. Variants of KEX2 and Kex2p may have atleast 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO:23 and SEQ ID NO: 24, respectively. KEX2 may or may not beoverexpressed.

A preferred host cell, most preferably S. cerevisiae, overexpresses PD/1and/or ERO1 and comprises at least one nucleic acid encoding Kex2p.

The nucleotide sequences encoding the Not4 protein, or homolog thereof,and desired proteins can be prepared using any mutagenesis procedureknown in the art, such as site-directed mutagenesis, synthetic geneconstruction, semi-synthetic gene construction, random mutagenesis,shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare polypeptides.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO95/17413; or WO95/22625. Other methods that can be usedinclude error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

A second aspect of the invention provides a culture of fungal host cellscontaining a polynucleotide sequence encoding a desired protein, such asa heterologous protein, characterised in that the fungal host cells havea modified, such as reduced, activity level of Not4 protein or homologthereof or a modified, such as reduced, expression level of Not4 proteinor homolog thereof. The fungal host cells according to the second aspectof the invention are as described for the first aspect of the invention.

Alternatively, the second aspect of the invention provides a culture offungal host cells containing a polynucleotide sequence encoding adesired protein, such as a heterologous protein, characterised in thatthe fungal host cells have an increased activity level of Not4 proteinor homolog thereof or an increased expression level of Not4 protein orhomolog thereof. The fungal host cells according to this alternativesecond aspect of the invention are as described for the first aspect ofthe invention. This may be useful for the production of a desiredprotein that is detrimental to the viability of the host.

A third aspect of the invention provides a method for producing adesired protein, such as a heterologous protein, from a fungal hostcell, the method comprising providing a fungal host cell according tothe first aspect of the invention or a culture according to the secondaspect of the invention and culturing the fungal host cell or culture toproduce the desired protein. The method may be used to modify theproduction yield of a desired polypeptide from a fungal host cell. Insome cases, it may be desirable to increase the production yield of someproteins. In other cases, it may be desirable to decrease the productionyield of some proteins, such as proteins that may be toxic to the hostcell.

The desired protein may or may not be secreted from the host cell, asecreted protein is preferred.

The host cells may be cultivated in a nutrient medium suitable forproduction of the desired protein using methods known in the art. Forexample, the cell may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermenters performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation maytake place in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). Preferred media include MW11D as described inExample 2. If the desired protein is secreted into the nutrient medium,the desired protein may be recovered directly from the medium. If thedesired protein is not secreted, it may be recovered from cell lysates.

The culturing may be at small or large scale, for example microtiterplate scale (e.g. from 10 to 500 microliter culture volume media), shakeflask scale (e.g. from 5 to 1000 milliliter (mL) culture volume), orfermenter or equivalent systems scale (e.g. at least from 5 mL culturevolume, more preferably at least 1, 2, 3, 4 or 5 liter (L), morepreferably at least 10, 50, 100 L, for example at least 500, 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000 L culturevolume).

The culturing may be at a pH suitable for the host cell. For S.cerevisiae, preferably the pH is from 5 to 7, for example from 5, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, or 6.9 to 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7. A preferred pH range isabout 6.0 to about 6.4.

The desired protein may be detected using methods known in the art thatare specific for the desired protein. These detection methods include,but are not limited to, use of specific antibodies, or high performanceliquid chromatography (HPLC).

A preferred HPLC is gel permeation HPLC (GP-HPLC). Suitable equipmentincludes a LC2010 HPLC system (Shimadzu) equipped with UV detectionunder Shimadzu VP7.3 client server software control. Injections of 75 μLmay be made onto a 7.8 mm id×300 mm length TSK G3000SWXL column (TosohBioscience), with a 6.0 mm id×40 mm length TSK SW guard column (TosohBioscience). Samples may be chromatographed in 25 mM sodium phosphate,100 mM sodium sulphate, 0.05% (w/v) sodium azide, pH 7.0 at 1 mL·min⁻¹,with a run time of 20 minutes. Samples may be quantified by UV detectionat 280 nm, by peak area, relative to a recombinant human albuminstandard of known concentration (e.g. 10 mg/mL) and corrected for theirrelative extinction coefficients.

Optionally, the method comprises recovering the desired protein, forexample isolating the desired protein from the host cell or host cellculture, e.g. cell media or cell lysate.

The desired protein may be recovered using methods known in the art. Forexample, the desired protein may be recovered from the nutrient mediumby conventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

Optionally, the method comprises purifying the desired protein. Thedesired protein may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure desired proteins.

In an alternative aspect, the desired protein is not recovered, butrather a host cell of the present invention expressing the desiredprotein is used as a source of the desired protein.

The step of purifying the desired protein (such as a desiredheterologous protein) from the cultured host cell or the culture mediumoptionally comprises cell immobilization, cell separation and/or cellbreakage, but always comprises at least one other purification stepdifferent from the step or steps of cell immobilization, separationand/or breakage.

Cell immobilization techniques, such as encasing the cells using calciumalginate bead, are known in the art. Similarly, cell separationtechniques, such as centrifugation, filtration (e.g. cross-flowfiltration), expanded bed chromatography and the like are known in theart.

Likewise, methods of cell breakage, including beadmilling, sonication,enzymatic exposure and the like are known in the art.

The at least one other purification step may be any other step suitablefor protein purification known in the art. For example purificationtechniques for the recovery of recombinantly expressed albumin have beendisclosed in: WO2010/128142, affinity purification using an albuminspecific ligand such as 2-chloro-4,6-di(2′-sulphoanilino)-S-triazine,WO92/04367, removal of matrix-derived dye; EP 464 590, removal ofyeast-derived colorants; EP319067, alkaline precipitation and subsequentapplication of the albumin to a lipophilic phase; and WO96/37515, U.S.Pat. No. 5,728,553 and WO00/44772, which describe complete purificationprocesses; all of which are incorporated herein by reference.

Desired proteins other than albumin may be purified from the culturemedium by any technique that has been found to be useful for purifyingsuch proteins.

Suitable methods include ammonium sulphate or ethanol precipitation,acid or solvent extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography, lectinchromatography, concentration, dilution, pH adjustment, diafiltration,ultrafiltration, high performance liquid chromatography (“HPLC”),reverse phase HPLC, conductivity adjustment and the like.

Optionally, the method may comprise purifying the isolated protein to acommercially or industrially acceptable level of purity. By commerciallyor industrially acceptable level of purity, we include the provision ofthe protein at a concentration of at least 0.01 g·L⁻¹, 0.02 g·L⁻¹, 0.03g·L⁻¹, 0.04 g·L⁻¹, 0.05 g·L⁻¹, 0.06 g·L⁻¹, 0.07 g·L⁻¹, 0.08 g·L⁻¹, 0.09g·L⁻¹, 0.1 g·L⁻¹, 0.2 g·L⁻¹, 0.3 g·L⁻¹, 0.4 g·L⁻¹, 0.5 g·L⁻¹, 0.6 g·L⁻¹,0.7 g·L⁻¹, 0.8 g·L⁻¹, 0.9 g·L⁻¹, 1 g·L⁻¹, 2 g·L⁻¹, 3 g·L⁻¹, 4 g·L⁻¹, 5g·L⁻¹, 6 g·L⁻¹, 7 g·L⁻¹, 8 g·L⁻¹, 9 g·L⁻¹, 10 g·L⁻¹, 15 g·L⁻¹, 20 g·L⁻¹,25 g·L⁻¹, 30 g·L⁻¹, 40 g·L⁻¹, 50 g·L⁻¹, 60 g·L⁻¹, 70 g·L⁻¹, 80 g·L⁻¹, 90g·L⁻¹, 100 g·L⁻¹, 150 g·L⁻¹, 200 g·L⁻¹, 250 g·L⁻¹, 300 g·L⁻¹, 350 g·L⁻¹,400 g·L⁻¹, 500 g·L⁻¹, 600 g·L⁻¹, 700 g·L⁻¹, 800 g·L⁻¹, 900 g·L⁻¹, 1000g·L⁻¹, or more. By commercially or industrially acceptable level ofpurity, we include the provision of the isolated protein in which othermaterial (for example, one or more (e.g. several) contaminants) arepresent at a level of less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%,1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, or 0.000001% and, mostpreferably at a level of 0%.

The protein may be provided at a concentration of at least 0.01 g·L⁻¹,0.02 g·L⁻¹, 0.03 g·L⁻¹, 0.04 g·L⁻¹, 0.05 g·L⁻¹, 0.06 g·L⁻¹, 0.07 g·L⁻¹,0.08 g·L⁻¹, 0.09 g·L⁻¹, 0.1 g·L⁻¹, 0.2 g·L⁻¹, 0.3 g·L⁻¹, 0.4 g·L⁻¹, 0.50=¹, 0.6 g·L⁻¹, 0.7 g·L¹, 0.8 g·L⁻¹, 0.9 g·L⁻¹, 1 g·L⁻¹, 2 0=¹, 3 g·L⁻¹,4 g·L⁻¹, 5 g·L⁻¹, 6 g·L⁻¹, 7 g·L⁻¹, 8 g·L⁻¹, 9 g·L⁻¹, 10 g·L⁻¹, 15g·L⁻¹, 20 g·L⁻¹, 25 g·L⁻¹, 30 g·L⁻¹, 40 g·L⁻¹, 50 g·L⁻¹, 60 g·L⁻¹, 70g·L⁻¹, 80 g·L⁻¹, 90 g·L⁻¹, 100 g·L⁻¹, 150 g·L⁻¹, 200 g·L⁻¹, 250 g·L⁻¹,300 g·L⁻¹, 350 g·L⁻¹, 400 g·L⁻¹, 500 g·L⁻¹, 600 g·L⁻¹, 700 g·L⁻¹, 800g·L⁻¹, 900 g·L⁻¹, 1000 g·L⁻¹, or more.

It is preferred that the desired protein is purified to achieve apharmaceutically acceptable level of purity. A protein has apharmaceutically acceptable level of purity if it is essentially pyrogenfree and can be administered in a pharmaceutically efficacious amountwithout causing medical effects not associated with the activity of theprotein.

Optionally, the method further comprises formulating the desired proteinwith a therapeutically acceptable carrier or diluent thereby to producea therapeutic product suitable for administration to a human or ananimal.

The resulting desired protein may, or may not, be used for any of itsknown utilities, which, in the case of albumin, include intra venous(i.v.) administration to patients to treat severe burns, shock and bloodloss, supplementing culture media, and as an excipient in formulationsof other proteins.

Although it is possible for a therapeutically, diagnostically,industrially, domestically or nutritionally useful desired proteinobtained by a process of the invention to be presented or administeredalone, it is preferable to present it as a formulation (such as apharmaceutical formulation, particularly in the case of therapeuticallyand/or diagnostically useful proteins), together with one or moreacceptable carriers or diluents. The carrier(s) or diluent(s) must be“acceptable” in the sense of being compatible with the desired proteinand, where the formulation is intended for administration to arecipient, then not deleterious to the recipient thereof. Typically, thecarriers or diluents will be water or saline which will be sterile andpyrogen free.

Optionally the thus formulated protein will be presented in a unitdosage form, such as in the form of a tablet, capsule, injectablesolution or the like.

Optionally, the method further comprises providing the desired proteinin unit dosage form.

A fourth aspect of the invention provides a method for increasing theyield of a desired protein (such as a heterologous protein) comprisingthe method according to the second aspect of the invention.

The fourth aspect of the invention also provides use of a host cellaccording to the first aspect of the invention or a culture according tothe second aspect of the invention to increase the yield of a desiredprotein (such as a heterologous protein).

Yield refers to the amount of product, for example desired protein, insolution, for example culture broth or cell lysis mixture. Yield may beexpressed in relative terms, e.g. the yield from a reference host strainbeing 100%. When comparing host strains, it is preferred that the yieldis measured under a defined set of conditions. Absolute yield may beexpressed as nanograms per microliter (ng/μL) or grams per liter (g/L).

Preferably, the yield of the desired protein is at least 2% higher thanthe yield from a reference fungal host cell such as a fungal host cellhaving a wild-type Not4 protein, such as SEQ ID NO: 2, more preferablyat least 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5,30, 35, 40, 45, or at least 50% higher. A preferred reference fungalhost cell has a Not4 protein of SEQ ID NO: 2.

The desired protein may be as described for the first aspect of theinvention, especially an albumin or variant, fragment and/or fusionthereof.

A fifth aspect of the invention provides a desired protein (such as aheterologous protein) produced by the method according to the second,third or fourth aspect of the invention.

The invention also provides a composition, such as a pharmaceuticalcomposition, comprising the desired protein of the fourth aspect of theinvention. The pharmaceutical composition may comprise one or morepharmaceutically acceptable carriers such as those approved by aregulatory authority such as the US Food and Drug Administration orEuropean Medicines Agency. The invention further provides a method oftreating a patient comprising administering an effective amount of thepharmaceutical composition to the patient.

A sixth aspect of the invention provides a method of preparing a fungalhost cell according to the first aspect of the invention or a cultureaccording to the second aspect of the invention. The method comprisesgenetically modifying a (parent) fungal host cell to modify theresultant Not4 protein or homolog thereof, to modify, e.g. reduce, theactivity level of Not4 protein or homolog thereof, to modify a NOT4 geneor homolog thereof or a control sequence thereof or to modify theexpression level of a NOT4 gene or homolog thereof. Mutations, deletionsand modification of activity and/or expression levels may be asdescribed for the first, second, and third aspects of the invention.Methods for engineering host cells are known in the art.

A seventh aspect of the invention provides a Not4 protein, or homologthereof, comprising at least 70% identity to SEQ ID NO: 2 and a mutationat a position corresponding to one or more position selected from 426,427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,469 or 470 of SEQ ID NO: 2, preferably a position selected from (a) 429,430, 434, or 437; (b) 463, 464 or 466; or (c) 442, 445, 447 or 452. Amutation at a position corresponding to position 429 of SEQ ID NO: 2 isparticularly preferred.

The Not4 protein according to the seventh aspect of the invention may beas described in relation to the first aspect of the invention.Preferably the Not4 protein comprises or consists of SEQ ID NO: 4. TheNot4 protein of the seventh aspect of the invention may or may not be anisolated protein.

An eighth aspect of the invention provides a polynucleotide encoding aNot4 variant of the present invention, such as a variant of SEQ ID NO: 2which results in a lower level of Not4 protein expression, or homologthereof, or a lower activity level of Not4 protein, or homolog thereof,than a host cell encoding a wild-type Not4 protein such as SEQ ID NO: 2,or homolog thereof. Such Not4 proteins are described in the first tosixth aspects of the invention.

A preferred polynucleotide encodes a Not4 protein with the mutationF4291 (SEQ ID NO: 4), an example of such a polynucleotide sequence isprovided by SEQ ID NO: 3.

For example, the present invention also relates to nucleic acidconstructs comprising a polynucleotide encoding a Not4 variant of thepresent invention operably linked to one or more control sequences thatdirect the expression of the coding sequence in a suitable host cellunder conditions compatible with the control sequences. Suitable controlsequences are described in the first to sixth aspects of the invention.

The polynucleotide may be located on a vector or in the genome of thehost cell.

Consequently, the present invention also relates to recombinant vectorscomprising a polynucleotide encoding a Not4 variant of the presentinvention, a promoter, and transcriptional and translational stopsignals. The invention also relates to vectors comprising apolynucleotide encoding Not4 and one or more (e.g. several) controlsequences which cause the level of Not4 or Not4 activity to be modified,for example reduced. The various nucleotide and control sequences may bejoined together to produce a recombinant vector that may include one ormore convenient restriction sites to allow for insertion or substitutionof the polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant vector may be any vector (e.g., a plasmid or virus) thatcan be conveniently subjected to recombinant DNA procedures and canbring about expression of the polynucleotide. The choice of the vectorwill typically depend on the compatibility of the vector with the hostcell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermits selection of transformed, transfected, transduced, or the likecells. A selectable marker is a gene, the product of which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a desired protein.An increase in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant vectors of the present invention are known to oneskilled in the art (see, e.g., Sambrook et al., 1989, Molecular Cloning,A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).

Preferred Embodiments

1. A fungal host cell having:

-   -   a. a modified Not4 protein or homolog thereof, or    -   b. a modified activity level or expression level of Not4 protein        or homolog thereof, or    -   c. a modified NOT4 gene or homolog thereof, or    -   d. a modified level of expression of NOT4 gene or homolog        thereof.        2. The fungal host cell of embodiment 1, wherein the modified        level is a reduced level.        3. The fungal host cell of embodiment 1, wherein the modified        level is an increased level.        4. The fungal host cell of any preceding embodiment, wherein the        modified level is relative to the level to a reference fungal        host cell, such as:    -   a. a fungal host cell in which the Not4 protein or homolog        thereof is a wild-type Not4 protein or homolog thereof,    -   b. a fungal host cell in which the Not4 protein comprises or        consists of SEQ ID NO: 2,    -   c. S. cerevisiae S288C or    -   d. S. cerevisiae DXY1.        5. The fungal host cell according to any preceding embodiment,        comprising a nucleotide sequence encoding a desired protein such        as heterologous protein.        6. The fungal host cell according to embodiment 5 in which the        desired protein is selected from albumin, a monoclonal antibody,        an etoposide, a serum protein (such as a blood clotting factor),        antistasin, a tick anticoagulant peptide, transferrin,        lactoferrin, endostatin, angiostatin, collagens, immunoglobulins        or immunoglobulin-based molecules or fragment of either (e.g. a        Small Modular ImmunoPharmaceutical™ (“SMIP”) or dAb, Fab′        fragments, F(ab′)2, scAb, scFv or scFv fragment), a Kunitz        domain protein (such as those described in WO03/066824, with or        without albumin fusions), interferons, interleukins, IL-10,        IL-11, IL-2, interferon α (alpha) species and sub-species,        interferon β (beta) species and sub-species, interferon γ        (gamma) species and sub-species, leptin, CNTF, CNTF_(Ax15),        IL-1-receptor antagonist, erythropoietin (EPO) and EPO mimics,        thrombopoietin (TPO) and TPO mimics, prosaptide, cyanovirin-N,        5-helix, T20 peptide, T1249 peptide, HIV gp41, HIV gp120,        urokinase, prourokinase, tPA, hirudin, platelet derived growth        factor, parathyroid hormone, proinsulin, insulin, glucagon,        glucagon-like peptides such as exendin-4, GLP-1 or GLP-2,        insulin-like growth factor, calcitonin, growth hormone,        transforming growth factor β (beta), tumour necrosis factor,        G-CSF, GM-CSF, M-CSF, FGF, coagulation factors in both pre and        active forms, including but not limited to plasminogen,        fibrinogen, thrombin, pre-thrombin, pro-thrombin, von        Willebrand's factor, alpha₁-antitrypsin, plasminogen activators,        Factor VII, Factor VIII, Factor IX, Factor X and Factor XIII,        nerve growth factor, LACI, platelet-derived endothelial cell        growth factor (PD-ECGF), glucose oxidase, serum cholinesterase,        aprotinin, amyloid precursor protein, inter-alpha trypsin        inhibitor, antithrombin III, apo-lipoprotein species, Protein C,        Protein S, a metabolite, an antibiotic, or a variant or fragment        of any of the above.        7. The fungal host cell according to embodiment 5 or 6 in which        the desired protein comprises or consists of an albumin,        variant, fragment and/or fusion thereof.        8. The fungal host cell according to embodiment 7 in which the        albumin or variant, fragment and/or fusion thereof has at least        70% identity to SEQ ID NO: 6.        9. The fungal host cell according to embodiment 7 in which the        albumin or variant, fragment and/or fusion thereof has at least        75, 80, 85, 90, 91, 92, 93, 95, 96, 97, 98 or 99% identity to        SEQ ID NO: 6.        10. The fungal host cell according to embodiment 9 in which the        albumin or variant, fragment and/or fusion thereof has at least        70% identity to SEQ ID NO: 6, preferably at least 75, 80, 85,        90, 91, 92, 93, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 6,        and comprises a A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T,        V, W or Y at a position corresponding to K573 of SEQ ID NO: 6.        11. The fungal host cell according to embodiment 10 in which        albumin or variant, fragment and/or fusion thereof comprises a        P, H, W or Y at a position corresponding to K573 of SEQ ID NO:        6.        12. The fungal host cell according to embodiment 11 in which the        albumin variant, fragment and/or fusion thereof has at least 98%        identity to SEQ ID NO: 6, and comprises a P at a position        corresponding to K573 of SEQ ID NO: 6.        13. The fungal host cell according to any of embodiments 5 to 12        in which the fusion comprises a fusion partner which is not        albumin or a variant or a fragment or fusion thereof.        14. The fungal host cell according to any of embodiments 7 to 13        in which the fusion comprises a fusion partner selected from        monoclonal antibody, an etoposide, a serum protein (such as a        blood clotting factor), antistasin, a tick anticoagulant        peptide, transferrin, lactoferrin, endostatin, angiostatin,        collagens, immunoglobulins or immunoglobulin-based molecules or        fragment of either (e.g. a Small Modular ImmunoPharmaceutical™        (“SMIP”) or dAb, Fab′ fragments, F(ab′)2, scAb, scFv or scFv        fragment), a Kunitz domain protein (such as those described in        WO03/066824, interferons, interleukins, IL-10, IL-11, IL-2,        interferon α (alpha) species and sub-species, interferon β        (beta) species and sub-species, interferon γ (gamma) species and        sub-species, leptin, CNTF, CNTF_(Ax15), IL-1-receptor        antagonist, erythropoietin (EPO) and EPO mimics, thrombopoietin        (TPO) and TPO mimics, prosaptide, cyanovirin-N, 5-helix, T20        peptide, T1249 peptide, HIV gp41, HIV gp120, urokinase,        prourokinase, tPA, hirudin, platelet derived growth factor,        parathyroid hormone, proinsulin, insulin, glucagon,        glucagon-like peptides such as exendin-4, GLP-1 or GLP-2,        insulin-like growth factor, calcitonin, growth hormone,        transforming growth factor β (beta), tumour necrosis factor,        G-CSF, GM-CSF, M-CSF, FGF, coagulation factors in both pre and        active forms, including but not limited to plasminogen,        fibrinogen, thrombin, pre-thrombin, pro-thrombin, von        Willebrand's factor, alpha₁-antitrypsin, plasminogen activators,        Factor VII, Factor VIII, Factor IX, Factor X and Factor XIII,        nerve growth factor, LACI, platelet-derived endothelial cell        growth factor (PD-ECGF), glucose oxidase, serum cholinesterase,        aprotinin, amyloid precursor protein, inter-alpha trypsin        inhibitor, antithrombin III, apo-lipoprotein species, Protein C,        Protein S, a metabolite, an antibiotic, or a variant or fragment        of any of the above.        15. The fungal host cell according to embodiment 13 or 14 in        which the fusion partner comprises or consists of a        glucagon-like protein or analog thereof.        16. The fungal host cell according to embodiment 15 in which the        fusion partner comprises or consists of SEQ ID NO: 10 or SEQ ID        NO: 11.        17. The fungal host cell according any of embodiments 5 to 16 in        which the desired protein comprises or consists of SEQ ID NO:        12.        18. The fungal host cell according to any of embodiments 2 to 17        in which the modified activity level or expression level of Not4        protein or homolog thereof is relative to the activity level or        expression level of Not4 protein or homolog thereof of a parent        fungal host cell such as a wild-type fungal host cell.        19. The fungal host cell according to embodiment 18, in which        the activity level of Not4 protein or homolog thereof is reduced        to no more than 90% of the activity level of Not4 protein or        homolog thereof of the parent fungal host cell.        20. The fungal host cell according to embodiment 19, in which        the activity level of Not4 protein or homolog thereof is reduced        to no more than 80% of the activity level of Not4 protein or        homolog thereof of the parent fungal host cell.        21. The fungal host cell according to embodiment 20, in which        the activity level of Not4 protein or homolog thereof is reduced        to no more than 70% of the activity level of Not4 protein or        homolog thereof of the parent fungal host cell.        22. The fungal host cell according to embodiment 21, in which        the activity level of Not4 protein or homolog thereof is reduced        to no more than 60% of the activity level of Not4 protein or        homolog thereof of the parent fungal host cell.        23. The fungal host cell according to embodiment 22, in which        the activity level of Not4 protein or homolog thereof is reduced        to no more than 50% of the activity level of Not4 protein or        homolog thereof of the parent fungal host cell.        24. The fungal host cell according to embodiment 23, in which        the activity level of Not4 protein or homolog thereof is reduced        to no more than 40% of the activity level of Not4 protein or        homolog thereof of the parent fungal host cell.        25. The fungal host cell according to embodiment 24, in which        the activity level of Not4 protein or homolog thereof is reduced        to no more than 30% of the activity level of Not4 protein or        homolog thereof of the parent fungal host cell.        26. The fungal host cell according to embodiment 25, in which        the activity level of Not4 protein or homolog thereof is reduced        to no more than 20% of the activity level of Not4 protein or        homolog thereof of the parent fungal host cell.        27. The fungal host cell according to embodiment 26, in which        the activity level of Not4 protein or homolog thereof is reduced        to substantially 0% of the activity level of Not4 protein or        homolog thereof of the parent fungal host cell.        28. The fungal host cell according to any preceding embodiment,        in which the host cell lacks a functional NOT4 gene or homolog        thereof or functional Not4 protein or homolog thereof.        29. The fungal host cell according to any preceding embodiment,        in which the host cell lacks a NOT4 gene or homolog thereof or        Not4 protein or homolog thereof.        30. The fungal host cell according to any preceding embodiment,        in which the NOT4 gene or homolog thereof or Not4 protein or        homolog thereof is mutated to alter the interaction of the Not4        protein or homolog thereof with a Not1 protein or homolog        thereof.        31. The fungal host cell according to any preceding embodiment        in which the Not4 protein or homolog thereof comprises a        mutation at position corresponding to a position selected from        426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438,        439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451,        452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464,        465, 466, 467, 468, 469 or 470 of SEQ ID NO: 2.        32. The fungal host cell according to any preceding embodiment        in which the position is selected from a position corresponding        to 429, 430, 434, or 437 of SEQ ID NO: 2.        33. The fungal host cell according to any preceding embodiment        in which the position is selected from a position corresponding        to 463, 464 or 466 of SEQ ID NO: 2.        34. The fungal host cell according to any preceding embodiment        in which the position is selected from a position corresponding        to 442, 445, 447 or 452 of SEQ ID NO: 2.        35. The fungal host cell according to any of embodiments 32 to        34 in which the mutation is a substitution, preferably to a        non-conserved amino acid.        36. The fungal host cell according to embodiment 31, 32 or 35 in        which the mutation at a position corresponding to position 429        of SEQ ID NO: 2 is a substitution to A, C, D, E, G, H, I, K, L,        M, N, P, Q, R, S, T, V, W or Y, preferably to G, A, V, L or I,        more preferably to I, L or V, most preferably to I.        37. The fungal host cell according to embodiment 31, 32 or 35 in        which the mutation at a position corresponding to position 429        of SEQ ID NO: 2 is a substitution from an aromatic amino acid to        an aliphatic amino acid.        38. The fungal host cell according to embodiment 36 or 37 in        which the Not4 protein comprises or consists of SEQ ID NO: 4.        39. The fungal host cell according to any preceding embodiment        comprising a modified NOT4 gene, for example a polynucleotide        encoding SEQ ID NO: 4.        40. The fungal host cell according to any preceding embodiment        in which the host cell lacks a NOT4 gene or homolog thereof or        Not4 protein or homolog thereof.        41. The fungal host cell according to any preceding embodiment,        in which one or more of the following chaperones are        overexpressed: AHA1, CCT2, CCT3, CCT4, CCT5, CCT6, CCT7, CCT8,        CNS1, CPR3, CPRE, ER01, EUG1, FM01, HCH1, HSP10, HSP12, HSP104,        HSP26, HSP30, HSP42, HSP60, HSP78, HSP82, JEM1, MDJ1, MDJ2,        MPD1, MPD2, PDI1, PFD1, ABC1, APJ1, ATP11, ATP12, BTT1, CDC37,        CPR7, HSC82, KAR2, LHS1, MGE1, MRS11, NOB1, ECM10, SSA1, SSA2,        SSA3, SSA4, SSC1, SSE2, SIL1, SLS1, ORM1, ORM2, PER1, PTC2,        PSE1, UBI4 and HAC1 or a truncated intronless HAC1, T/M9, PAM18,        TCP1 or a variant thereof.        42. The fungal host cell according to any preceding embodiment        in which KEX2, or a variant or fragment thereof, is expressed or        overexpressed.        43. The fungal host cell according to embodiment 41 or 42 in        which PDI1 or a variant thereof is overexpressed or ERO1 or a        variant thereof is overexpressed.        44. The fungal host cell according to embodiment 41 or 42 in        which PDI1 or a variant thereof is overexpressed and ER01 or a        variant thereof are overexpressed.        45. The fungal host cell according to embodiment 41 or 42 in        which PDI1 or a variant thereof is overexpressed and KEX2 or a        variant thereof is expressed or overexpressed.        46. The fungal host cell according to embodiment 41 or 42 in        which ER01 or a variant thereof is overexpressed and KEX2 or a        variant thereof is expressed or overexpressed.        47. The fungal host cell according to any of embodiments 41 to        46 in which PDI1 or a variant thereof is overexpressed and ER01        or a variant thereof is overexpressed and KEX2 or a variant        thereof is expressed or overexpressed.        48. The fungal host cell according to any preceding embodiment        in which the fungal host is a yeast or a filamentous fungus.        49. The fungal host cell, according to any preceding embodiment,        in which the host cell is a Saccharomyces, Candida, Hansenula,        Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or        Yarrowia.        50. The fungal host cell according to embodiment 49 in which the        Saccharomyces is a Saccharomyces carlsbergensis, Saccharomyces        cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,        Saccharomyces kluyveri, Saccharomyces norbensis, or        Saccharomyces oviformis, preferably Saccharomyces cerevisiae.        51. A culture of fungal host cells containing a polynucleotide        sequence encoding a desired protein, such as a heterologous        protein, characterised in that the fungal host cells have a        reduced activity level of Not4 protein or homolog thereof.        52. The culture of fungal host cells of embodiment 51 in which        the host cells are as defined in any of embodiments 1 to 50.        53. A method for producing a desired protein, such as a        heterologous protein, from a fungal host cell comprising:    -   (i) providing a fungal host cell according to any of embodiments        1 to 50 or a culture according to embodiment 51 or 52,    -   (ii) culturing the fungal host cell or culture to produce the        desired protein,    -   (iii) optionally recovering the desired protein,    -   (iv) optionally purifying the desired protein,    -   (v) optionally formulating the desired protein with a        therapeutically acceptable carrier or diluent thereby to produce        a therapeutic product suitable for administration to a human or        an animal, and    -   (vi) optionally providing the desired protein in unit dosage        form.        54. A method for increasing the yield of a desired protein (such        as a heterologous protein) comprising:    -   (i) providing a fungal host cell (such as a yeast or a        filamentous fungus) having:        -   a. a modified Not4 protein or homolog thereof, or        -   b. a modified level of activity (preferably reduced) of Not4            protein or homolog thereof, or        -   c. a modified NOT4 gene or homolog thereof, or        -   d. a modified level of expression (preferably reduced) of            NOT4 gene or homolog thereof.    -   (ii) culturing the host cell to produce the desired protein, and    -   (iii) optionally recovering the desired protein,    -   (iv) optionally purifying the desired protein,    -   (v) optionally formulating the desired protein with a        therapeutically acceptable carrier or diluent thereby to produce        a therapeutic product suitable for administration to a human or        an animal, and    -   (vi) optionally providing the desired protein in unit dosage        form.        55. The method according to embodiment 53 or 54 in which the        yield of the desired protein is at least 2% higher than the        yield from a reference fungal host cell such as a fungal host        cell having a wild-type Not4 protein, such as SEQ ID NO: 2.        56. The method according to embodiment 55 in which the yield is        at least 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 15, 17.5, 20, 22.5, 25,        27.5, 30, 35, 40, 45, or at least 50% higher than the yield from        a reference fungal host cell.        57. The method according to embodiment 55 or 56 in which the        yield of the desired protein is at least 2% higher than the        yield from a reference fungal host cell such as a fungal host        cell having Not4 protein of SEQ ID NO: 2.        58. The method according to embodiment 57 in which the yield is        at least 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 15, 17.5, 20, 22.5, 25,        27.5, 30, 35, 40, 45, or at least 50% higher than the yield from        a reference fungal host cell.        59. The method according to any of embodiments 53 to 58 in which        the desired protein is selected from albumin, a monoclonal        antibody, an etoposide, a serum protein (such as a blood        clotting factor), antistasin, a tick anticoagulant peptide,        transferrin, lactoferrin, endostatin, angiostatin, collagens,        immunoglobulins or immunoglobulin-based molecules or fragment of        either (e.g. a Small Modular ImmunoPharmaceutical™ (“SMIP”) or        dAb, Fab′ fragments, F(ab′)2, scAb, scFv or scFv fragment), a        Kunitz domain protein (such as those described in WO03/066824,        with or without albumin fusions), interferons, interleukins,        IL-10, IL-11, IL-2, interferon α (alpha) species and        sub-species, interferon β (beta) species and sub-species,        interferon γ (gamma) species and sub-species, leptin, CNTF,        CNTF_(Ax15), IL-1-receptor antagonist, erythropoietin (EPO) and        EPO mimics, thrombopoietin (TPO) and TPO mimics, prosaptide,        cyanovirin-N, 5-helix, T20 peptide, T1249 peptide, HIV gp41, HIV        gp120, urokinase, prourokinase, tPA, hirudin, platelet derived        growth factor, parathyroid hormone, proinsulin, insulin,        glucagon, glucagon-like peptides such as exendin-4, GLP-1 or        GLP-2, insulin-like growth factor, calcitonin, growth hormone,        transforming growth factor β (beta), tumour necrosis factor,        G-CSF, GM-CSF, M-CSF, FGF, coagulation factors in both pre and        active forms, including but not limited to plasminogen,        fibrinogen, thrombin, pre-thrombin, pro-thrombin, von        Willebrand's factor, alpha₁-antitrypsin, plasminogen activators,        Factor VII, Factor VIII, Factor IX, Factor X and Factor XIII,        nerve growth factor, LACI, platelet-derived endothelial cell        growth factor (PD-ECGF), glucose oxidase, serum cholinesterase,        aprotinin, amyloid precursor protein, inter-alpha trypsin        inhibitor, antithrombin III, apo-lipoprotein species, Protein C,        Protein S, a metabolite, an antibiotic, or a variant or fragment        of any of the above.        60. The method according to any of embodiments 53 to 59 in which        the desired protein comprises or consists of an albumin or        variant, fragment and/or fusion thereof.        61. The method according to embodiment 60 in which the albumin        or variant, fragment and/or fusion thereof has at least 70%        identity to SEQ ID NO: 6.        62. The method according to embodiment 61 in which the albumin        or variant, fragment and/or fusion thereof has at least 75, 80,        85, 90, 91, 92, 93, 95, 96, 97, 98 or 99% identity to SEQ ID NO:        6.        63. The method according to embodiment 62 in which the albumin        or variant, fragment and/or fusion thereof has at least 70%        identity to SEQ ID NO: 6, preferably at least 75, 80, 85, 90,        91, 92, 93, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 6, and        comprises an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V,        W or Y at a position corresponding to K573 of SEQ ID NO: 6.        64. The method according to embodiment 63 in which the albumin        or variant, fragment and/or fusion thereof comprises a P, H, W        or Y at a position corresponding to K573 of SEQ ID NO: 6.        65. The method according to embodiment 64 in which the albumin        or variant, fragment and/or fusion thereof has thereof has at        least 98% identity to SEQ ID NO: 6, and comprises a P at a        position corresponding to K573 of SEQ ID NO: 6.        66. The method according to any of embodiments 53 to 65 in which        the fusion comprises a fusion partner which is a not albumin or        a variant, fragment and/or fusion thereof.        67. The method according to any of embodiments 53 to 66 in which        the fusion comprises a fusion partner selected from monoclonal        antibody, an etoposide, a serum protein (such as a blood        clotting factor), antistasin, a tick anticoagulant peptide,        transferrin, lactoferrin, endostatin, angiostatin, collagens,        immunoglobulins or immunoglobulin-based molecules or fragment of        either (e.g. a Small Modular ImmunoPharmaceutical™ (“SMIP”) or        dAb, Fab′ fragments, F(ab′)2, scAb, scFv or scFv fragment), a        Kunitz domain protein (such as those described in WO03/066824,        interferons, interleukins, IL-10, IL-11, IL-2, interferon α        (alpha) species and sub-species, interferon β (beta) species and        sub-species, interferon γ (gamma) species and sub-species,        leptin, CNTF, CNTF_(Ax15), IL-1-receptor antagonist,        erythropoietin (EPO) and EPO mimics, thrombopoietin (TPO) and        TPO mimics, prosaptide, cyanovirin-N, 5-helix, T20 peptide,        T1249 peptide, HIV gp41, HIV gp120, urokinase, prourokinase,        tPA, hirudin, platelet derived growth factor, parathyroid        hormone, proinsulin, insulin, glucagon, glucagon-like peptides        such as exendin-4, GLP-1 or GLP-2, insulin-like growth factor,        calcitonin, growth hormone, transforming growth factor β (beta),        tumour necrosis factor, G-CSF, GM-CSF, M-CSF, FGF, coagulation        factors in both pre and active forms, including but not limited        to plasminogen, fibrinogen, thrombin, pre-thrombin,        pro-thrombin, von Willebrand's factor, alpha₁-antitrypsin,        plasminogen activators, Factor VII, Factor VIII, Factor IX,        Factor X and Factor XIII, nerve growth factor, LACI,        platelet-derived endothelial cell growth factor (PD-ECGF),        glucose oxidase, serum cholinesterase, aprotinin, amyloid        precursor protein, inter-alpha trypsin inhibitor, antithrombin        III, apo-lipoprotein species, Protein C, Protein S, a        metabolite, an antibiotic, or a variant or fragment of any of        the above.        68. The method according to embodiment 67 in which the fusion        partner comprises or consists of a glucagon-like protein or        analog thereof.        69. The method according to embodiment 68 in which the fusion        partner comprises or consists of SEQ ID NO: 10 or SEQ ID NO: 11.        70. The method according to any of embodiments 53 to 69 in which        the desired protein comprises or consists of SEQ ID NO: 12.        71. The method according to any of embodiments 53 to 70 in which        the host cell is cultured at a scale of at least 1 L.        72. The method according to embodiment 71 in which the host cell        is cultured at a scale of at least 2 L.        73. The method according to embodiment 72 in which the host cell        is cultured at a scale of at least 5 L.        74. The method according to embodiment 73 in which the host cell        is cultured at a scale of at least 10 L.        75. The method according to embodiment 74 in which the host cell        is cultured at a scale of at least 1000 L.        76. The method according to embodiment 75 in which the host cell        is cultured at a scale of at least 5000 L.        77. The method according to any of embodiments 53 to 76 in which        the desired protein is secreted from the fungal host cell.        78. The method according to embodiment 77 in which the desired        protein results from an immature protein comprising a signal        peptide.        79. The method according to embodiment 78 in which the signal        peptide comprises or consists of SEQ ID NO: 13, SEQ ID NO: 14,        SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ        ID NO: 20, SEQ ID NO: 35 or SEQ ID NO: 36 or a signal peptide        comprising the pentapeptide motif of SEQ ID NO: 16.        80. The method according to embodiment 79 in which the signal        peptide comprises or consists of SEQ ID NO: 15.        81. The method according to embodiment 79 in which the signal        peptide comprises or consists of SEQ ID NO: 20.        82. The method according to embodiment 79 in which the signal        peptide comprises or consists of SEQ ID NO: 36.        83. The method according to any of embodiments 53 to 77 in which        the desired protein is intracellular.        84. A desired protein (such as a heterologous protein) produced        by the method according to any of embodiments 53 to 83.        85. The desired protein according to embodiment 84 for        prophylaxis, therapy or diagnosis.        86. A composition, such as a pharmaceutical composition,        comprising the desired protein according to embodiment 84 or 85        and a pharmaceutically acceptable carrier.        87. A method of treatment comprising administering the desired        protein of embodiment 84 or 85 or the composition of embodiment        86 to a patient.        88. A method of preparing a fungal host cell according to any of        embodiments 1 to 50 or a culture according to embodiment 51 or        52, the method comprising genetically modifying a (parent)        fungal host cell to reduce the activity level of Not4 protein or        homolog thereof.        89. Use of a means to reduce the activity level of Not4 protein        or homolog thereof in a fungal host cell to increase the yield        of a desired protein (such as a heterologous protein) from the        fungal host cell, for example: by mutating or deleting the NOT4        gene, thus resulting a mutated Not4 protein or homolog thereof        or complete absence of Not4 protein or homolog thereof; by        removing or changing the open reading frame of the gene, by        mutating or changing control sequences of the NOT4 gene such as        a promoter sequence and/or a terminator sequence; by blocking or        reducing transcription of the NOT4 gene for example by        introducing suitable interfering RNA such as antisense mRNA, by        introducing, controlling or modifying suitable transcriptional        activator genes or by introducing an agent which blocks activity        level of Not4 protein or homolog thereof..        90. A Not4 protein or homolog thereof, comprising at least 70%        identity to SEQ ID NO: 2 and a mutation at a position        corresponding to one or more position selected from 426, 427,        428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,        441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453,        454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466,        467, 468, 469 or 470 of SEQ ID NO: 2.        91. The Not4 protein, or homolog thereof, according to        embodiment 90 in which the position is selected from a position        corresponding to 429, 430, 434, or 437 of SEQ ID NO: 2.        92. The Not4 protein, or homolog thereof, according to        embodiment 90 in which the position is selected from a position        corresponding to 463, 464 or 466 of SEQ ID NO: 2.        93. The Not4 protein, or homolog thereof, according to        embodiment 90 in which the position is selected from a position        corresponding to 442, 445, 447 or 452 of SEQ ID NO: 2.        94. The Not4 protein, or homolog thereof, according to any of        embodiments 90 to 93 in which the mutation is a substitution,        preferably to a non-conserved amino acid.        95. The Not4 protein, or homolog thereof, according to        embodiment 90 or 91 in which the mutation at a position        corresponding to position 429 of SEQ ID NO: 2 is a substitution        to A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y,        preferably to G, A, V, L or I, more preferably to I, L or V,        most preferably to I.        96. The Not4 protein, or homolog thereof, according to        embodiment 90 or 91 in which the mutation at a position        corresponding to position 429 of SEQ ID NO: 2 is a substitution        from an aromatic amino acid to an aliphatic amino acid.        97. The Not 4 protein, or homolog thereof, according to any of        embodiments 90, 91, 95 or 96 comprising or consisting of SEQ ID        NO: 4.

EXAMPLES Example 1: Mutation of the Saccharomyces cerevisiae NOT4 Gene

S. cerevisiae DP9 has the genotype cir⁰ MATa, leu2-3, leu2-112 ubc4 ura3yap3::URA3 lys2 hsp150:LYS2 with PDI1, URA3 and Ylplac211 integrated atthe PDI1 locus (Finnis et al, 2010, Microbial Cell Factories 9: 87). Theinventors observed that S. cerevisiae DP9 (when transformed with analbumin-encoding plasmid) was able to produce recombinant human albuminat a higher yield than predecessor strains e.g. S. cerevisiae DB1.Characterisation of S. cerevisiae DP9 revealed a single polynucleotidepolymorphism (SNP) in the NOT4 gene. In order to identify whether or notthis SNP contributed to the improved protein yield of S. cerevisiae DP9,the SNP (T1285A, SEQ ID NO: 3) was reverted to the wild-type (i.e. T atposition 1285, SEQ ID NO: 1) as described below. Consequently, themutant Not4 protein (1429, SEQ ID NO: 4) was also reverted to wild-type(F429, SEQ ID NO: 2).

The Saccharomyces cerevisiae NOT4 gene is located on chromosome V. TheSNP (T1285A) in the mutant NOT4 gene was reverted to wild type by theprocess of integrating a fragment into the NOT4 locus, changing base1285A to T, thus reverting the mutant Not4 protein (1 at position 429,SEQ ID NO: 4) to wild-type Not4 protein (F at position 429, SEQ ID NO:2).

This was achieved by first amplifying, by PCR, a suitable selectionmarker (KanMX) with mutagenic single stranded DNA primers which modifiedthe 5′ and 3′ ends of the KanMX gene so as to include DNA sequencesidentical to regions downstream of the NOT4 open reading frame (SEQ IDNO: 25)). The PCR primers were Primer A and Primer B, KanMX confersresistance to geneticin (G418).

Primer A: (SEQ ID NO: 26)5′-CCGTTTATAACGAAATGCAAGAAAAAAAAATCTCACCCATTTTTTTAAACCTTTGACGTGGAAAGGTATCTGGGAAAGGTATCTGGCTAATGAATAATGCCGTACGCTGCAGGTCG-3′ Primer B (SEQ ID NO: 27)5′-ATATATCATGATGATTATTTTCTATGAATTAGTCATTCTTGCAGCGCTGACGCTTTCATACGTTGTAACGAGTAAATAGACTATACTGGTATATGCTATGATCGATGAATTCGAGCTCG-3′

A PCR reaction was performed to amplify the KanMX gene from the plasmidpDB5438 (FIG. 1. Conditions were as follows: 100 ng plasmid pDB5438, 0.5μM of each primer, initial denaturation for 30 seconds at 98° C., then35 cycles with 98° C. for 10 seconds, annealing at 63° C. for 30seconds, extension at 72° C. for 1.5 minutes, followed by a finalextension at 72° C. for 4 minutes, and cooling to 4° C., using anApplied Biosystems 2720 Thermal Cycler and a NEB Q5 Hot StartHigh-Fidelity DNA Polymerase PCR kit (M0493S), total reaction volume 50μL, according to the manufacturer's instructions.

The product, 5′-NOT4 3′UTR-KanMX-NOT4 3′UTR-3′, was analysed by gelelectrophoresis and was found to be of the expected size, approximately1.6 kb. The amplified PCR product was purified using a QIAGEN QIAquickPCR Purification kit according to the manufacturer's instructions. Thepurified product was used to transform a S. cerevisiae strain which waswild-type for NOT4 (i.e. SEQ ID NO: 1). Transformation was done using aSigma Yeast Transformation kit according to the manufacturer'sinstructions, except after the step where the transformation mix iscentrifuged, the pellet was re-suspended in 1 mL YEPD medium, and thentransferred to a 30 mL Sterilin tube containing 3 mL YEPD. YEPD (g/L):10 g Bacto™ Yeast Extract Technical, 20 g Bacto™ Peptone, 20 g Glucose.

The tube was incubated for 16 hours at 30° C. with shaking (200 rpm).The Sterilin tube was centrifuged at 3,000 rpm for 5 minutes and thesupernatant decanted. Then the pellet was re-suspended in 500 μl 1Msorbitol. About 150 μl was then plated onto freshly prepared G418 agarplates (300 μg/ml G418 final concentration) and incubated face-down at30° C. for five days. The G418 agar plates were prepared as following:0.17 g yeast nitrogen base (without (NH₄)₂SO₄), 0.1 g glutamic acid(monosodium salt, Sigma G-1626), 0.069 g CSM-Leu powder, 100 ml H₂O(sterile water for irrigation—nonpyrogenic, hypotonic) and 1 g Bactoagar were added to a 200 mL autoclaved glass bottle and mixed. Thebottle was heated in a steamer for one hour and then cooled to 55° C. ina water bath. 0.6 ml 50 mg/ml Geneticin (G418) and 4 mL sterile 50%dextrose (w/v) were added and mixed. Aliquots of the mixture were pouredinto petri dishes to set.

Genomic DNA was extracted from G418 resistant transformants and used asa template in a second PCR, using primers MBP260 and MBP266, to amplifya 5′-NOT4-NOT4 3′UTR-KanMX-NOT4 3′UTR-3′ fragment (SEQ ID NO: 28)containing the 3′ part of the NOT4 gene, the NOT4 3′ UTR, the KanMXgene, and downstream sequence.

Primer MBP260: (SEQ ID NO: 29) 5′-TGCAAGATGTATAGCTCAGG-3′ Primer MBP266:(SEQ ID NO: 30) 5′-TGCAAATCCTGCTATGGTGG-3′

The PCR materials, method and conditions were as described above. Theproduct, 5′-NOT4-NOT4 3′UTR-KanMX-NOT4 3′UTR-3′, was analysed by gelelectrophoresis and was found to be of the expected size, approximately3.3 kb. The amplified PCR product was purified using a QIAGEN QIAquickPCR Purification kit according to the manufacturer's instructions. Thepurified product was used to transform DP9 [pDB2305] using thetransformation method described above. S. cerevisiae DP9 is a straincontaining the NOT4 SNP (T1285A, F4291)). pDB2305 is a plasmid encodinghuman albumin (FIG. 3). The outgrowth and selection on G418 agar plateswere as described above. Genomic DNA was extracted from resistantcolonies, and PCR was used to amplify an about 4.5 kb fragment usingprimers MBP269 and MBP287. The same PCR kit and conditions were usedexcept the cycling steps were changed to 98° C. for 10 seconds,annealing at 62° C. for 20 seconds, and extension at 72° C. for 2.5minutes.

Primer MBP269: (SEQ ID NO: 31) 5′-ATAAAATCACCTGGCATTACG-3′Primer MBP287: (SEQ ID NO: 32) 5′-CAACAGTTGGATCACAGTGG-3′

The products were cleaned as described above. A Life Technologies BigDyeTerminator v3.1 Cycle Sequencing kit was used for the sequencing theproducts according to the manufacturer's instructions, using 50 μL totalreaction volumes, with SOng of the cleaned products as template and 4 μLof 1 μM primers (MBP274 and MBP282. The conditions were as following:Initial denaturation 96° C. 1 min. Then 25 cycles: Denaturation 96° C.10 seconds, annealing 50° C. 5 seconds, elongation 60° C. 4 minutes, andthen cooling to 4° C. The sequencing reactions were precipitated andresuspended in HiDi (Applied Biosystems) and analysed on an AppliedBiosystems 3130xl Genetic Analyser.

Primer MBP274: (SEQ ID NO: 33) 5′-CTCTGGGCCATCATACTACC-3′ Primer MBP282:(SEQ ID NO: 34) 5′-GTTGCTGCTGAATAGGAACC-3′

The sequencing analysis showed that three transformants had the wildtype T at position 1285 (F429), this strain was named PRM5. Twotransformants still had the A at position 1285 (1429), this strain wasnamed PSM7. Three PRM5 transformants and two PSM7 transformants werecultured in a 48-well microtiter plate (MTP), containing 0.5 mL BMMD(0.17% (w/v) yeast nitrogen base without amino acid and ammoniumsulphate (Difco), 37.8 mM ammonium sulphate, 36 mM citric acid, 126 mMdisodium hydrogen orthophosphate pH6.5, 2% (w/v) glucose, adjusted to pH6.5 with NaOH) in each well (six replicates for each transformant). TheMTP was incubated at 30° C. in a humidity chamber with shaking (200 rpm)for 48 hours. Then 50 μL cell culture from each well was transferredinto wells in a new 48-well MTP containing 0.45 mL BMMD in each well.The new MTP was incubated at 30° C. in a humidity chamber with shaking(200 rpm) for 96 hours.

The supernatant was isolated by centrifugation and recombinant albuminproductivity was determined by GP-HPLC analysis using a LC2010 HPLCsystem (Shimadzu) equipped with UV detection under Shimadzu VP7.3 clientserver software control. Injections of 75 μL were made onto a 7.8 mmid×300 mm length TSK G3000SWXL column (Tosoh Bioscience), with a 6.0 mmid×40 mm length TSK SW guard column (Tosoh Bioscience). Samples werechromatographed in 25 mM sodium phosphate, 100 mM sodium sulphate, 0.05%(w/v) sodium azide, pH 7.0 at 1 mL·min-1, with a run time of 20 minutes.Samples were quantified by UV detection at 280 nm, by peak area,relative to a recombinant human albumin standard of known concentration(10 mg/mL) and corrected for their relative extinction coefficients.

As shown in Table 2, the presence of the SNP resulted in an 18% increasein average albumin yield.

TABLE 2 Albumin productivity in PRM5 [pDB2305] and PSM7 [pDB2305] PRM5[pDB2305] PSM7 [pDB2305] Albumin 100% ± 8.3 118% ± 10.1 (relative yield)P value (t-test): 8.06E−07

The work was repeated in a further S. cerevisiae strain. Briefly, thesame SNP was reverted to wild-type in S. cerevisiae BXP10 and the yieldof albumin from BXP10 (containing the SNP, i.e. BSM6 [pDB2244]) wascompared with the yield of albumin from the BXP10 strain with the SNPconverted to wild-type (BRM4 [pDB2244]). BXP10 has the genotype MATa,leu2-3, leu2-122, can1, pra1, ubc4, ura3, yap3::URA3, lys2,hsp150::LYS2, and pmt1::URA3.

As shown by Table 3, the presence of the SNP resulted in an 8% increasein albumin yield (6 replicates for each strain).

TABLE 3 Albumin productivity in BRM4 [pDB2244] and BSM6 [pDB2244] BRM4[pDB2244] BSM6 [pDB2244] Albumin 100% ± 8.9 108% ± 8.1 (relative yield)P value (t-test): 0.0348

Example 2: Mutation of the Saccharomyces cerevisiae NOT4 Gene Enhancedthe Production of Recombinant Protein at 10 L Scale

The productivity of S. cerevisiae PRM5 [pDB2305] and PSM7 [pDB2305] wasassessed by growth in 10 L fermenter (Wigley et al (2007) GeneticEngineering News. 27(2):40-42). The fermentation was performed asdescribed in Example 1 of WO97/33973 using MW11D medium, except thatWonderware Supervisory Control and Data Acquisition software was usedinstead of MFCS software, prior to use the fermentation vessel was alsosubjected to a citric acid wash, the trace element stock comprisedNa₂MoO₄.2H₂O instead of Na₂MoO₄.5H₂O, the initial pH was adjusted withammonia solution (specific gravity 0.901) to pH 6.0 to 6.4, initialintroduction of sterile air into the vessel was at about 1.0 wm (i.e.1.0 liter) instead of 0.5 wm, during fermentation the airflow wasincreased in one step instead of two to maintain an airflow ofapproximately 1.0 vvm, the specific growth rate was approximately0.06h⁻¹ and the exponential constant (K) was kept at 0.06.

The recombinant albumin productivity was determined by GP.HPLC against arecombinant albumin standard. The recombinant albumin productivity ofPSM7 [pDB2305] under these conditions was calculated to be about 13%higher than the productivity of PRM5 [pDB2305], measured under identicalconditions (Table 4).

TABLE 4 Albumin productivity in PRM7 [pDB2305] and PSM7 [pDB2305] at 10L scale PRM5 [pDB2305] PSM7 [pDB2305] Albumin 100% ± 6 113% ± 7(relative yield) P value (t-test): 0.009. N = 4

The work was repeated in S. cerevisiae BXP10 at 10 L scale, and theyield of albumin from BXP10 (containing the SNP, i.e. BSM6 [pDB2305])was compared with the yield of albumin from the BXP10 strain with theSNP converted to wild-type (BRM4 [pDB2305]). As shown by Table 5, thepresence of the SNP resulted in a 15% increase in albumin yield (2replicates for each strain).

TABLE 5 Albumin productivity in BRM4 [pDB2244] and BSM6 [pDB2244] at 10L scale BRM4 [pDB2244] BSM6 [pDB2244] Albumin 100% ± 6 115% ± 5(relative yield) P value (t-test): 0.038

Example 3: Deletion of the Saccharomyces cerevisiae NOT4 Gene Enhancedthe Production of Recombinant Protein

The NOT4 gene was deleted in a Saccharomyces cerevisiae MT302/28B cir⁰(MATα, leu2, pep4-3, Finnis et al 1993, Eur. J. Biochem, 212: 201-210),containing plasmid pDB2244 which encodes human serum albumin. Thedeletion was achieved by replacing the NOT4 gene with the marker KanMX.Consequently, the resultant strain (MT302/2B Δnot4) was unable toproduce any Not4 protein.

Strain MT302/28B and strain MT302/28B Δnot4 were then cultured (eightreplicates for each strain) and the albumin productivity determined asdescribed in Example 1. As shown in Table 6, deletion of NOT4 resultedin a 61% increase in albumin yield.

TABLE 6 Albumin productivity in Strain MT302/28B [pDB2244] and StrainMT302/28B ΔNOT4 [pDB2244] MT302/28B MT302/28B Δnot4 [pDB2244] [pDB2244]Albumin 100% ± 0.9 161% ± 5.6 (relative yield) P value (t-test): 5.9E−09

Example 4: Mutation of the Saccharomyces cerevisiae NOT4 Gene Enhancedthe Expression of an Albumin Fusion Protein (Albumin-IL-1Ra) and scFv(vHvL)-FLAG

The proteins being expressed in this example were (a) IL-1Ra geneticallyfused to the C-terminal of human serum albumin (SEQ ID NO: 38) and (b)the scFv, FITC8 (Evans et al 2010, Protein Expression and Purification73:113-124, including references 16 and 17, all incorporated herein byreference) with a FLAG tag (DYKDDDDK) at its C-terminal (SEQ ID NO: 40).

In preparation for expression of albumin-IL-1Ra, plasmid pDB3936 (FIG.7.) was cut with restriction enzymes Acc65I and BamHI and plasmidpDB5912 (containing an albumin-IL-1 Ra expression cassette) was cut withenzymes NsiI and PvuI. A plasmid map for pDB5912 is provided in FIG. 6.,the DNA sequence encoding IL-1Ra is shown in SEQ ID NO: 37. Therestriction enzymes and buffers were from New England Biolabs. Bothplasmid digests were purified using a Qiagen PCR purification kitfollowing the manufacturer's instructions.

The 4 strains, PRM5 [pDB2305], PSM7 [pDB2305], BRM4 [pDB2244] and BSM6[pDB2244], were cultured in shake flasks in YEPD media and subcultured 3times in order to cure them of the plasmid (pDB2305 or pDB2244).Dilutions of the final cultures were plated onto YEPD and then singlecolonies from these plates were patched onto YEPD. The YEPD patches weretransferred to BMMD plates and incubated at 30^(Q)C; a lack of growth onBMMD identified the cells which had been cured of plasmid. The curedyeast strains were each transformed, using the Sigma YeastTransformation kit according to the manufacturer's instructions, withplasmid pDB3029 (for expression of scFv (vHvL)-FLAG (a plasmid map forpDB3029 is provided in FIG. 5., the DNA sequence encoding scFv-FLAG isshown in SEQ ID NO: 39), or with the purified restriction digests ofpDB3936 and pDB5912 (for expression of albumin-IL-1 RA from thegap-repaired plasmid pDB3936:GR:pDB5912). The cells were plated ontoBMMD and incubated for 5 days at 30° C. Six transformants of each werecultured in a 48 well MTP containing 0.5 ml BMMD per well. The plate wasincubated for 48 hours at 30° C. and 200 rpm in a humidity chamber. Thisplate was then subcultured by transferring 50 μl of each culture into450 μl BMMD in a new plate. This plate was incubated for 96 hours.

The supernatant was isolated by centrifugation and recombinant proteinproductivity (albumin-IL-1Ra or ScFv) was determined by GP-HPLC, as inExample 1.

As shown in Table 7 and Table 8, the presence of the SNP (F4291)resulted in an increase in yield of albumin-IL-1Ra. In the DP9 derivedstrains, the yield was 8% higher in the strain containing the SNP inNOT4 (PSM7), compared to the strain with wild-type NOT4 (PRM5) (Table7). In the BXP10 derived strains, the yield was 24% higher in the straincontaining the SNP in NOT4 (BSM6), compared to the strain with wild-typeNOT4 (BRM4) (Table 8).

TABLE 7 Albumin-IL-1Ra productivity in PRM5 [pDB3936:GR:pDB5912] andPSM7 [pDB3936:GR:pDB5912] PRM5 PSM7 [pDB3936:GR:pDB5912][pDB3936:GR:pDB5912] Albumin-IL-1Ra 100% +/− 5 108% +/− 6 (relativeyield) P value (t-test): P = 0.008

TABLE 8 Albumin-IL-1Ra productivity in BRM4 [pDB3936:GR:pDB5912] andBSM6 [pDB3936:GR:pDB5912] BRM4 BSM6 [pDB3936:GR:pDB5912][pDB3936:GR:pDB5912] Albumin-IL-1Ra 100% +/− 10 124% +/− 9 (relativeyield) P value (t-test): 0.002

As shown in Table 9 and Table 10, the presence of the SNP (F4291)resulted in an increase in yield of ScFv-FLAG. In the DP9 derivedstrains, the yield was 14% higher in the strain containing the SNP inNOT4 (PSM7), compared to the strain with wild-type NOT4 (PRM5) (Table9). In the BXP10 derived strains, the yield was 19% higher in the straincontaining the SNP in NOT4 (BSM6), compared to the strain with wild-typeNOT4 (BRM4) (Table 10).

TABLE 9 ScFv (vHvL)-FLAG productivity in PRM5 [pDB3029] and PSM7[pDB3029 PRM5 [pDB3029] PSM7 [pDB3029] ScFv 100% +/− 10 114% +/− 10(relative yield) P value (t-test): p = 0.004

TABLE 10 ScFv (vHvL)-FLAG productivity in BRM4 [pDB3029] and BSM6[pDB3029] BRM4 [pDB3029] BSM6 [pDB3029] ScFv 100% +/− 5 119% +/− 4(relative yield) P value (t-test): p = 1.65E−05

1. A fungal host cell having: a. a modified Not4 protein or homologthereof, or b. a modified activity level of Not4 protein or homologthereof, or c. a modified NOT4 gene or homolog thereof, or d. a modifiedlevel of expression of NOT4 gene, or homolog thereof.
 2. The fungal hostcell of claim 1, wherein the modified level is a reduced level.
 3. Thefungal host cell of claim 2, wherein the reduced level is relative tothe level to a reference fungal host cell, such as a fungal host cell inwhich the Not4 protein comprises or consists of SEQ ID NO:
 2. 4. Thefungal host cell according to claim 1, which is a yeast or a filamentousfungus.
 5. The fungal host cell according to claim 1, comprising anucleotide sequence encoding a desired protein such as heterologousprotein, such as a serum protein, preferably an albumin or variant,fragment and/or fusion thereof.
 6. The fungal host cell according toclaim 1, in which the Not4 protein comprises a mutation at positioncorresponding to a position selected from 426, 427, 428, 429, 430, 431,432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445,446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459,460, 461, 462, 463, 464, 465, 466, 467, 468, 469 or 470 of SEQ ID NO: 2,preferably a position selected from 429, 430, 434, or
 437. 7. The fungalhost cell according to claim 6 in which the mutation at a positioncorresponding to position 429 of SEQ ID NO: 2 is a substitution to A, C,D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y, preferably to G, A,V, L or I, more preferably to I, L or V, most preferably to I.
 8. Thefungal host cell according to claim 6 in which the mutation at aposition corresponding to position 429 of SEQ ID NO: 2 is F429I.
 9. Thefungal host cell according to claim 1, comprising a polynucleotideencoding SEQ ID NO:
 4. 10. The fungal host cell according to claim 1,which lacks a NOT4 gene, or homolog thereof, or Not4 protein, or homologthereof.
 11. The fungal host cell according to claim 1, in which thehost cell is a Saccharomyces such as Saccharomyces cerevisiae.
 12. Amethod for producing a desired protein, such as a heterologous protein,from a fungal host cell, said method comprising: (i) providing a fungalhost cell according to claim 1, (ii) culturing the fungal host cell or aculture thereof to produce the desired protein, (iii) optionallyrecovering the desired protein, (iv) optionally purifying the desiredprotein, (v) optionally formulating the desired protein with atherapeutically acceptable carrier or diluent thereby to produce atherapeutic product suitable for administration to a human or an animal,and (vi) optionally providing the desired protein in unit dosage form.13. A method for increasing the yield of a desired protein (such as aheterologous protein) comprising: (i) providing a fungal host cell (suchas a yeast or a filamentous fungus) having: a. a modified Not4 proteinor homolog thereof, or b. a modified level of activity (preferablyreduced) of Not4 protein or homolog thereof, or c. a modified NOT4 geneor homolog thereof, or d. a modified level of expression (preferablyreduced) of NOT4 gene, or homolog thereof, (ii) culturing the host cellto produce the desired protein, and (iii) optionally recovering thedesired protein, (iv) optionally purifying the desired protein, (v)optionally formulating the desired protein with a therapeuticallyacceptable carrier or diluent thereby to produce a therapeutic productsuitable for administration to a human or an animal, and (vi) optionallyproviding the desired protein in unit dosage form.
 14. The methodaccording to claim 12, in which the yield of the desired protein is atleast 2% higher than the yield from a reference fungal host cell such asa fungal host cell having Not4 protein of SEQ ID NO:
 2. 15. The methodaccording to claim 12, in which the desired protein is an albumin orvariant, fragment and/or fusion thereof.
 16. The method according toclaim 12, in which the host cell is cultured at a scale of at least 5 L.17. (canceled)